KR101316095B1 - Composition for preventing and treating of neuropathic pain containing ginsenoside Rb１ and Rg３，Compound K，or saponin extract from Panax ginseng as an effective ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating neuropathic pain containing ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract as an active ingredient, specifically, ginsenosides Rb1 and Rg3, Compound K, or Ginseng-derived saponin extracts have been shown to inhibit the pain caused by central nerve damage, peripheral nerve damage, and inflammation. These pain-inhibiting effects are mediated by estrogen receptors and also pain-related factors in the spinal cord microglial cells. It acts by inhibiting expression.

Description

Composition for preventing and treating neuropathic pain containing ginsenoside Rb1 and Rg3, Compound K ， or saponin, containing ginsenosides R1 and R3, CO3, CO3, or ginseng-derived saponin extract as an active ingredient extract from Panax ginseng as an effective ingredient}

The present invention is a composition for preventing and treating neuropathic pain containing ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract as an active ingredient, and for preventing and improving neuropathic pain containing the active ingredient. It is about food.

In general, pain is one of the physiological reactions that appear as a life-threatening or defense mechanism against strong external stimuli and is known to play an important role in protecting the body from danger. Pain is divided into nocieptive pain due to tissue damage and neuropathic pain, which does not involve tissue damage but is caused by a nervous system reaction. Invasive pain is known to be closely related to pain, accompanied by an inflammatory response due to tissue damage, and generally disappears as the damaged area heals. Neuropathic pain is a chronic neurological disease caused by damage to the nervous system due to various causes such as trauma, inflammation, ischemic damage, or metabolites (Gilron et al., 2006; Barn, 2006). Neuropathic pain is characterized by accompanying symptoms such as spontaneous pain that occurs without any stimulus and hyperalgesia or allodynia caused by external stimulation. Hyperalgesia is defined as excessive abnormal pain response to noxious stimuli, and allodynia is known as pain response caused by harmless stimuli (Woolf and Mannion, 1999). Neuropathic pain is known to be difficult to relieve pain with conventional painkillers. Neuropathic pain inhibitors include anticonvulsants, such as Gabapentin and Pregabalin, which act on calcium channels, and anticonvulsants, such as Carbamazepine, which act on sodium channels; Serotonin Norepinephrine Reuptake Inhibitor (SNRI) family antidepressants; Four classes of opioids, used only in emergencies, are used clinically.

In particular, complete or partial spinal nerve injury causes neuropathic pain within a few months after injury in the majority of patients with spinal cord injury (Beric, 1997; Christensen and Hulsebosch, 1997; Yezierski. 1996 Yu et. al., 2003) ultimately developing chronic neuropathic pain that persists for a long time (Richards et al., 1980). The pain caused after spinal cord injury mainly occurs in three areas: the above-level pain, which is the pain in the upper part of the injury area, the at-level pain, the pain in the area close to the injury area or the same dermatome. It is divided into below-level pain, which is pain at the lower part of the site (Siddall et al., 1997; Siddall and cousins, 1997). These pains, which occur after spinal cord injury, vary from mild numbness to extreme pain that attempts suicide, and often lead to chronic pain, which bothers patients so that their daily lives are impossible. Moreover, neuropathic pain is not only a pain itself, but also a mental stress such as sleep disorders, anxiety and depression, which not only threaten the quality of life of the patient, but also incur significant treatment costs (for patients with spinal cord injuries). 25 million won per year, about 1 billion won per lifetime), which has become a serious social and economic problem beyond patients and families (Cairns et al., 1996; Segatore, 1994).

Little is known about the mechanisms of pain induced above the injury site, whereas the mechanisms of injury and pain underneath the injury site have been focused on the goal of treating pain after spinal nerve injury. (Hulsebosch. 2002; Yezierski. 2004). Inflammatory responses are known to play a pivotal role in controlling pathologies of acute and chronic spinal cord injury (Bligh, 1992), which are the primary causes of secondary injury following spinal cord injury (Bligh, 1992). Is known to mediate neuropathic pain, especially below-level pain, after spinal cord injury (Hains and Wanxman, 2006; Zhao et al., 2007). Therefore, the control of the inflammatory response through the inhibition of microglial cell activation is a key strategy for the treatment of spinal cord injury and pain control. However, studies on inflammatory response control mechanisms and microglial cell activation are very early and intensive studies are needed.

As mentioned above, drugs used for neuropathic pain are mostly used in the clinic, but each class of drugs has similar side effects, so the combination may worsen the side effects in some patients. There is a problem that the effect is low. In particular, despite the development of various analgesics or analgesic methods to date, 75% of patients with neuropathic pain still report severe pain, but the mechanism of causing neuropathic pain is not clearly established. Since painkillers do not adequately cure neuropathic pain, there is no toxicity and side effects, and more pain inhibitors or therapeutic agents are required.

Accordingly, the inventors of the present invention ginsenoside Rb1 and Rg3, and ginseng-derived saponin extract isolated from ginseng-derived saponin extract, and the composition containing Compound K as an active ingredient in the rat nerve model after injury to the central nerve, respectively When administered, mechanical allodynia and heat hyperalgesia were reduced, and this pain-inhibiting effect occurred through the estrogen receptor and suppressed pain by suppressing the expression of p-p38MAPK, pERK, and pJNK, which are pain related factors in the spinal cord. This invention was completed by confirming that the effect to make is shown.

An object of the present invention is Panax ginseng ginseng ) Saponin extract derived from or to provide a composition for preventing and treating neuropathic pain, or health food containing saponin isolated from the active ingredient.

Still another object of the present invention is to provide a neuropathic pain prevention and treatment composition or health food containing any one or more selected from the group consisting of ginsenosides Rb1 and Rg3 and Compound K as an active ingredient.

In order to achieve the above object, the present invention ginseng ( Panax ginseng ) provides a composition for preventing and treating neuropathic pain containing saponin extract or isolated saponin as an active ingredient.

The present invention also provides a composition for preventing and treating neuropathic pain, which contains the compound represented by the following [Formula 1] as an active ingredient.

In addition, the present invention ginseng ( Panax ginseng ) Provides a health food for preventing and improving neuropathic pain containing saponin extract derived from the ginseng or isolated saponin as an active ingredient.

In addition, the present invention provides a neuropathic pain prevention and improvement health food containing the compound represented by [Formula 1] as an active ingredient.

Wherein, R1 is Glc _{2 -} is Glc or H;

R2 is Glc ₆ -Glc or H or Glc.

Ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts according to the present invention, respectively, have the effect of suppressing pain caused by inflammation as well as central nerve damage and peripheral nerve damage. Mediated, and also acts by inhibiting the expression of inflammation-related factors of the microglial cells in the spinal cord, at least one component selected from the group consisting of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention The composition containing the as an active ingredient can be usefully used for the prevention and treatment of pain.

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4);
도 2의 B는 중추 신경병증성 통증 동물모델에 본 발명에 따른 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 3일 동안 투여하면서, 기계적 이질통(mechanical allodynia) 테스트를 수행한 결과이다;
○ Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=6);
∇ Minocycline; 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=6);
□ Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
◇ Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
△ Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4);
도 2의 C는 중추 신경병증성 통증 동물모델에 본 발명에 따른 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 3일 동안 투여하면서, 열 통각과민(heat hyperalgesia) 테스트를 수행한 결과이다;
○ Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=6);
∇ Minocycline; 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=6);
□ Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
◇ Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
△ Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4);
도 2의 D는 BBB, 기계적 이질통, 및 열통각과민 테스트별 최고점 대비 그룹의 평균값을 배분율로 나타낸 그래프이다;
1 Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=6);
2 Minocycline: 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=6);
3 Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
4 Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
5 Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);
6 Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4).
도 3은 인삼유래 사포닌 추출물의 농도에 따른 중추 신경병증성 통증의 억제효과를 나타낸 그래프이다:
도 3의 A는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 본 발명에 따른 인삼유래 사포닌 추출물을 농도별로 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 기계적 이질통 테스트를 수행한 결과를 나타낸 그래프이다;
△ Vehicle: 래트 1마리당 10㎕의 생리식염수를 척수강내로 투여한 군(n=4);
◇ Total saponin 20 ㎍: 래트 1마리당 20 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
□ Total saponin 50 ㎍: 래트 1마리당 50 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
▽ Total saponin 100 ㎍: 래트 1마리당 100 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
○ Total saponin 200 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
도 3의 B는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 본 발명에 따른 인삼유래 사포닌 추출물중 Rb1만을 농도별로 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 기계적 이질통 테스트를 수행한 결과를 나타내 그래프이다;
◇ Vehicle:래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군(n=4);
□ Rb1 10 ㎍: 래트 1마리당 10 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=3);
∇ Rb1 25 ㎍: 래트 1마리당 25 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=4);
○ Rb1 50 ㎍: 래트 1마리당 50 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=4);
도 3의 C는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 본 발명에 따른 인삼유래 사포닌 추출물을 농도별로 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 열 통각과민 테스트를 수행한 결과를 나타내 그래프이다;
△ Vehicle: 래트 1마리당 10㎕의 생리식염수를 척수강내로 투여한 군(n=4);
◇ Total saponin 20 ㎍: 래트 1마리당 20 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
□ Total saponin 50 ㎍: 래트 1마리당 50 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
▽ Total saponin 100 ㎍: 래트 1마리당 100 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
○ Total saponin 200 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물을 척수강내로 투여한 군(n=4);
도 3의 D는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 본 발명에 따른 인삼유래 사포닌 추출물중 Rb1만을 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 열 통각과민 테스트를 수행한 결과를 나타내 그래프이다;
◇ Vehicle: 래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군(n=4);
□ Rb1 10 ㎍: 래트 1마리당 10 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=3);
∇ Rb1 25 ㎍: 래트 1마리당 25 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=4);
○ Rb1 50 ㎍: 래트 1마리당 50 ㎍의 진세노사이드 Rb1을 척수강내로 투여한 군(n=4).
도 4는 에스트로겐 수용체의 중추 신경병증성 통증감소의 억제 효과를 나타낸 그래프이다:
도 4의 A는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 인삼유래 사포닌 추출물과 에스트로겐 길항제를 각각, 또는 함께 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 기계적 이질통 테스트를 수행한 결과를 나타낸 그래프이다;
○ Total saponin 200 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물을 단독으로 척수강내로 투여한 군(n=4);
∇ Total saponin 200 ㎍ + ICI 50 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물과 에스트로겐 길항제 ICI 50 ㎍을 척수강내에 함께 투여한 군(n=4);
□ ICI 50 ㎍: 래트 1마리당 50 ㎍의 에스트로겐 길항제를 단독으로 척수강내에 투여한 군(n=4);
◇ Vehicle:래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군 (n=4);
도 4의 B는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 Rb1과 에스트로겐 길항제를 각각, 또는 함께 척수강내로 투여한 후, 1시간, 2시간 및 4시간에서 기계적 이질통 테스트를 수행한 결과를 나타낸 그래프이다;
○ Rb1 50 ㎍: 래트 1마리당 50 ㎍의 인삼유래 사포닌 추출물 Rb1을 단독으로 척수강내로 투여한 군(n=4);
∇ Rb1 50 ㎍ + ICI 50 ㎍: 래트 1마리당 50 ㎍의 인삼유래 사포닌 추출물 Rb1과 에스트로겐 길항제 ICI 50 ㎍을 척수강내에 함께 투여한 군(n=4);
□ ICI 50 ㎍: 래트 1마리당 50 ㎍의 에스트로겐 길항제 ICI를 단독으로 척수강내에 투여한 군(n=4);
◇ Vehicle: 래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군 (n=4);
도 4의 C는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후 만성통증이 생긴 동물모델에 인삼유래 사포닌 추출물과 에스트로겐 길항제를 각각, 또는 함께 척수강내로 투여한 후 1시간, 2시간 및 4시간에서 열 통각과민 테스트를 수행한 결과를 나타낸 그래프이다;
○ Total saponin 200 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물을 단독으로 척수강내로 투여한 군(n=4);
∇ Total saponin 200 ㎍ + ICI 50 ㎍: 래트 1마리당 200 ㎍의 인삼유래 사포닌 추출물과 에스트로겐 길항제 ICI 50 ㎍을 척수강내에 함께 투여한 군(n=4);
□ ICI 50 ㎍: 래트 1마리당 50 ㎍의 에스트로겐 길항제를 단독으로 척수강내에 투여한 군(n=4);
◇ Vehicle:래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군(n=4);
도 4의 D는 중추 신경병증성 통증 동물모델을 제조한 지, 4주가 경과한 후, 만성통증이 생긴 동물모델에, Rb1과 에스트로겐 길항제를 각각, 또는 함께 척수강내로 투여한 후, 1시간, 2시간 및 4시간에서 열 통각과민 테스트를 수행한 결과를 나타낸 그래프이다;
○ Rb1 50 ㎍: 래트 1마리당 50 ㎍의 진세노사이드 Rb1을 단독으로 척수강내로 투여한 군(n=4);
∇ Rb1 50 ㎍ + ICI 50 ㎍: 래트 1마리당 50 ㎍의 진세노사이드 Rb1과 에스트로겐 길항제 ICI 50 ㎍을 척수강내에 함께 투여한 군(n=4);
□ ICI 50 ㎍: 래트 1마리당 50 ㎍의 에스트로겐 길항제 ICI를 단독으로 척수강내에 투여한 군(n=4); 및
◇ Vehicle: 래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군(n=4).
도 5는 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물의 염증반응에 의해서 유발된 통증을 억제하는 효과를 나타낸 그래프이다:
도 5의 A는 염증반응을 유발시키는 포르말린을 주입 30분전에 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 투여하고 관찰되는 통증 행위(발을 핥거나 드는 행동)를 나타낸 그래프이다;
○ Vehicle: 래트 1마리당 10 ㎕의 생리식염수를 척수강내로 투여한 음성 대조군(n=8);
∇ Total saponin 200 ㎍: 래트 1마리당 인삼유래 사포닌 추출물 200 ㎍을 척수강내에 투여한 군(n=4);
□ Compound K 50 ㎍: 래트 1마리당 진세노사이드 Compound K 50 ㎍을 척수강내에 투여한 군(n=4);
◇ Rb1 50 ㎍: 래트 1마리당 진세노사이드 Rb1 50 ㎍을 척수강내에 투여한 군(n=3);
△ Rg3 50 ㎍: 래트 1마리당 진세노사이드 Rg3 50 ㎍을 척수강내에 투여한 군(n=3);
도 5의 B는 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 투여하고 30분 지난 래트에서 염증반응을 유발시키는 포르말린을 주입하자마자 관찰되는 통증 행위(발을 핥거나 드는 행동)를 0 ~ 10분, 10 ~ 60분대의 시간대로 나누어 관찰한 그래프이다;
Early phase: 포르말린 투여 후 0 ~ 10분 사이;
Late phase: 염증성 반응이 나타나는 10 ~ 60분 사이;
1 Vehicle: 10 ㎕ 생리식염수를 척수강내로 투여한 음성 대조군(n=8);
2 Total saponin 200 ㎍: 인삼유래 사포닌 추출물 200 ㎍/래트로 척수강내에 투여한 군(n=4);
3 Compound K 50 ㎍: 진세노사이드 Rb1의 대사산물인 Compound K를 50 ㎍/래트로 척수강내에 투여한 군(n=4);
4 Rb1 50 ㎍: 진세노사이드 Rb1 50 ㎍/래트로 척수강내에 투여한 군(n=4); 및
5 Rg3 50 ㎍: 진세노사이드 Rg3 50 ㎍/래트로 척수강내에 투여한 군(n=3).
도 6은 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물의 미세신경교세포의 활성억제 효과를 확인한 그림이다:
도 6의 A는 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 경구 투여한 후, 요추 4번 척수 후각부위의 라미나 1, 2번(lamina Ⅰ,Ⅱ)에서의 미세신경교세포의 활성억제 효과를 면역조직화학염색법을 이용하여 확인한 그림이다;
Vehicle: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=4);
Minocycline; 22.5 mg/kg의 미노사이클린을 하루에 2번 복강 투여한 양성 대조군(n=4);
Rb1: 11.5 mg/kg의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=4);
Compound K: 7 mg/kg의 진세노사이드 Compound K를 하루에 한번 경구 투여한 군(n=4);
Total saponin: 50 mg/kg의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=4);
Rg3: 11.5 mg/kg의 진세노사이드 Rg3을 하루에 한번 경구 투여한 군(n=4);
도 6의 B는 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 투여한 후, 요추 4번 척수 후각부위의 라미나 1, 2번(lamina Ⅰ,Ⅱ)에서의 활성화된 미세신경교세포와 비활성화된 미세신경교세포의 비율을 나타낸 그래프이다;
Vehicle: 1 ㎖의 생리식염수를 경구 투여한 음성 대조군으로서, 도 6의 A에 나타난 Vehicle 그림에서, 점선으로 둘러싸인 조직 부분의 총 67개의 세포수 중, 미세신경교세포의 활성(막대 그래프의 검정색)과 비활성(막대 그래프의 회색)을 나타낸다(n=4);
Minocycline; 22.5 mg/kg의 미노사이클린을 투여한 양성 대조군으로서, 도 6의 A에 나타난 Minocycline 그림에서, 점선으로 둘러싸인 조직 부분의 총 64개의 세포수 중, 미세신경교세포의 활성(막대그래프의 검정색)과 비활성(막대그래프의 회색)을 나타낸다(n=4);
Rb1: 11.5 mg/kg의 진세노사이드 Rb1을 투여한 군으로서, 도 6의 A에 나타난 Rb1 그림에서, 점선으로 둘러싸인 조직 부분의 총 61개의 세포수 중, 미세신경교세포의 활성(막대그래프의 검정색)과 비활성(막대그래프의 회색)을 나타낸다(n=4);
Compound K: 7 mg/kg의 진세노사이드 Compound K를 투여한 군으로서, 도 6의 A에 나타난 Compound K 그림에서, 점선으로 둘러싸인 조직 부분의 총 60개의 세포수 중, 미세신경교세포의 활성(막대그래프의 검정색)과 비활성(막대그래프의 회색)을 나타낸다(n=4);
Total saponins: 50 mg/kg의 인삼유래 사포닌 추출물을 투여한 군(n=4)으로서, 도 6의 A에 나타난 Total saponins 그림에서, 점선으로 둘러싸인 조직 부분의 총 62개의 세포수 중, 미세신경교세포의 활성(막대그래프의 검정색)과 비활성(막대그래프의 회색)을 나타낸다;
Rg3: 11.5 mg/kg의 진세노사이드 Rg3을 투여한 군으로서, 도 6의 A에 나타난 Rg3 그림에서, 점선으로 둘러싸인 조직 부분의 총 66개의 세포수 중, 미세신경교세포의 활성(막대 그래프의 검정색)과 비활성(막대 그래프의 회색)을 나타낸다(n=4).
도 7은 도 6의 A에서 진세노사이드 Rb1 및 Rg3, Compound K, 또는 인삼유래 사포닌 추출물을 투여한 후, 요추 4번 척수 후각부위의 라미나 1, 2번(lamina Ⅰ,Ⅱ)에서의 미세신경교세포의 활성화정도를 면역조직염색법을 이용하여 확인한 그림에서 점선으로 둘러싸인 조직 부위를 확대한 그림이다:
Vehicle: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=4);
Minocycline; 22.5 mg/kg의 미노사이클린을 하루에 2번 복강 투여한 양성 대조군(n=4);
Rb1: 11.5 mg/kg의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=4);
Compound K: 7 mg/kg의 진세노사이드 Compound K를 하루에 한번 경구 투여한 군(n=4);
Total saponin: 50 mg/kg의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=4); 및
Rg3: 11.5 mg/kg의 진세노사이드 Rg3을 하루에 한번 경구 투여한 군(n=4).
도 8은 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물의 p-p38MAPK의 활성에 미치는 영향을 확인하기 위하여, 중추 신경병증성 통증 동물모델의 요추 4번 척수를 분리해서 웨스턴 블랏을 수행한 결과이다:
도 8의 A는 중추 신경병증성 통증 동물모델에 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물을 투여한 후, p-p38MAPK의 활성을 확인하기 위하여 웨스턴 블랏을 수행한 결과를 나타내는 그림이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Min: 22.5 mg/kg의 미노사이클린을 하루에 두번 복강 투여한 군(n=5);
Total-sap: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5);
도 8의 B는 상기 웨스턴 블랏의 결과에서, 대조군의 발현량을 기준으로 p-p38MAPK의 발현량을 수치화한 그래프이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Minocycline: 22.5 mg/kg의 미노사이클린을 하루에 두번 복강 투여한 군(n=5);
Total saponins: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5); 및
*: P < 0.05.
도 9는 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물의 pERK(phosphorylated extracellular signal-regulated kinases)의 활성에 미치는 영향을 확인하기 위하여, 중추 신경병증성 통증 동물모델의 요추 4번 척수를 분리해서 웨스턴 블랏을 수행한 결과이다:
도 9의 A는 중추 신경병증성 통증 동물모델에 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물을 투여한 후, pERK의 활성을 확인하기 위하여 웨스턴 블랏을 수행한 결과를 나타내는 그림이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Min: 22.5 mg/kg의 미노사이클린을 하루에 두 번 복강 투여한 군(n=5);
Total-sap: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5);
도 9의 B는 상기 웨스턴 블랏의 결과에서, 대조군의 발현량을 기준으로 pERK의 발현량을 수치화한 그래프이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Minocycline: 22.5 mg/kg의 미노사이클린을 하루에 두번 복강 투여한 군(n=5);
Total saponins: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5); 및
*: P < 0.05.
도 10은 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물의 pJNK(phosphorylated c-Jun N-terminal kinases)의 활성에 미치는 영향을 확인하기 위하여, 중추 신경병증성 통증 동물모델의 요추 4번 척수를 분리해서 웨스턴 블랏을 수행한 결과이다:
도 10의 A는 중추 신경병증성 통증 동물모델에 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물을 투여한 후, pJNK의 활성을 확인하기 위하여 웨스턴 블랏을 수행한 결과를 나타내는 그림이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Min: 22.5 mg/kg의 미노사이클린을 하루에 두 번 복강 투여한 군(n=5);
Total-sap: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5);
도 10의 B는 상기 웨스턴 블랏의 결과에서, 대조군의 발현량을 기준으로 pJNK의 발현량을 수치화한 그래프이다;
Veh: 1 ㎖의 생리식염수를 하루에 한번 경구 투여한 음성 대조군(n=5);
Minocycline: 22.5 mg/kg의 미노사이클린을 하루에 두 번 복강 투여한 군(n=5);
Total saponins: 50 ㎎/㎏의 인삼유래 사포닌 추출물을 하루에 한번 경구 투여한 군(n=5);
Rb1: 11.5 ㎎/㎏의 진세노사이드 Rb1을 하루에 한번 경구 투여한 군(n=5); 및
*: P < 0.05.
도 11은 진세노사이드 Rb1, 및 인삼유래 사포닌 추출물의 말초 신경병증성 통증의 억제효과를 나타낸 그래프이다:
도 11의 A는 말초 신경병증성 통증 동물모델을 제조한 지, 2주가 경과한 후, 만성통증이 생긴 동물모델에, 본 발명에 따른 인삼유래 사포닌 추출물을 농도별로 척수강내로 투여한 후, 14, 15, 16일째에서 기계적 이질통 테스트를 수행한 결과를 나타내 그래프이다;
○ Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=4);
∇ Minocycline; 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=4);
□ Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
◇ Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
△ Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4);
도 11의 B는 말초 신경병증성 통증 동물모델을 제조한 지, 2주가 경과한 후, 만성통증이 생긴 동물모델에, 본 발명에 따른 인삼유래 사포닌 추출물을 농도별로 척수강내로 투여한 후, 14, 15, 16일째에서 냉 이질통(cold allodynia) 테스트를 수행한 결과를 나타내 그래프이다;
○ Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=4);
∇ Minocycline; 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=4);
□ Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
◇ Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
△ Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4);
도 11의 C는 말초 신경병증성 통증 동물모델을 제조한 지, 2주가 경과한 후, 만성통증이 생긴 동물모델에, 본 발명에 따른 인삼유래 사포닌 추출물을 농도별로 척수강내로 투여한 후, 14, 15, 16일째에서 온 이질통(warm allodynia) 테스트를 수행한 결과를 나타내 그래프이다;
○ Vehicle: 1 ㎖ 생리식염수를 투여한 음성 대조군(n=4);
∇ Minocycline; 22.5 mg/kg 미노사이클린을 투여한 양성 대조군(n=4);
□ Rb1: 11.5 mg/kg 진세노사이드 Rb1을 투여한 군(n=4);
◇ Compound K: 7 mg/kg의 진세노사이드 Rb1의 대사 산물을 투여한 군(n=4);
△ Total saponin: 50 mg/kg 인삼유래 사포닌 추출물을 투여한 군(n=4);

Rg3: 11.5 mg/kg 진세노사이드 Rg3을 투여한 군(n=4).1 is a diagram showing the location of nerve damage in the process of building animal models of peripheral neuropathic pain:
ossa coxae: two long bones;
S1: first sacral nerve (No. 1 Sacral nerve);
S2: second sacral nerve;
S3: third sacral nerve;
S4: fourth sacral nerve;
C1: first coccygeal nerve;
C2: second coccyx nerve; And
X: As the nerve injury position, specifically, the superior and inferior nerve positions are cut between the sacral nerves 1 and 2, and only the nerve 1 to the tail of the rat is damaged.
2 is 4 weeks after the preparation of a central neuropathic pain animal model, chronic pain occurs in the animal model, ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention After administration, the pain behavior response test was performed:
2A is a result of performing the BBB test while orally administering ginsenosides Rb1 and Rg3, Compound K, or ginseng derived saponin extract according to the present invention to a central neuropathic pain animal model;
Vehicle: negative control group (n = 6) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 6) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4);
2B is a result of performing a mechanical allodynia test while administering ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract for 3 days to a central neuropathic pain animal model. to be;
Vehicle: negative control group (n = 6) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 6) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4);
2C is a heat neuroal hypersensitivity (heat hyperalgesia) test while administering ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract for 3 days in a central neuropathic pain animal model The result is;
Vehicle: negative control group (n = 6) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 6) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4);
FIG. 2D is a graph showing the distribution of the mean values of the groups versus the peaks for the BBB, mechanical allodynia, and hyperalgesia test;
1 Vehicle: negative control group (n = 6) administered 1 ml physiological saline;
2 Minocycline: positive control (n = 6) administered 22.5 mg / kg minocycline;
3 Rb1: group administered 11.5 mg / kg ginsenoside Rb1 (n = 4);
4 Compound K: group administered with 7 mg / kg of ginsenoside Rb1 metabolite (n = 4);
5 Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);
6 Rg3: group administered 11.5 mg / kg ginsenoside Rg3 (n = 4).
Figure 3 is a graph showing the inhibitory effect of central neuropathic pain according to the concentration of ginseng-derived saponin extract:
FIG. 3A is a 1 hour, 2 hour after administration of the ginseng-derived saponin extract according to the present invention to the animal model in which chronic pain occurs after 4 weeks of preparation of a central neuropathic pain animal model. And a graph showing the results of performing a mechanical allodynia test at 4 hours;
Δ Vehicle: A group in which 10 μl of saline was injected into the spinal cavity per rat (n = 4);
◇ 20 μg of total saponin: group administered with 20 μg of ginseng-derived saponin extract per rat intranasally (n = 4);
□ 50 μg of total saponin: group administered with 50 μg of ginseng-derived saponin extract intraratically into the spinal cord (n = 4);
? Total saponin 100 μg: group administered 100 g of ginseng-derived saponin extract per rat intrathecalally (n = 4);
O Total saponin 200 μg: Group administered with 200 μg of ginseng-derived saponin extract per rat intrathecal (n = 4);
FIG. 3B is one hour after administration of the Rb1 in the ginseng-derived saponin extract according to the present invention to the animal model in which chronic pain occurs after 4 weeks after the preparation of the central neuropathic pain animal model. A graph showing the results of mechanical allodynia testing performed at 2 and 4 hours;
◇ Vehicle: negative control group (n = 4) administered 10 μl of saline per spinal cord per rat;
10 μg of Rb1: group in which 10 μg of ginsenoside Rb1 was administered intranasally (n = 3) per rat;
B 25 μg of Rb1: group administered with 25 μg of ginsenoside Rb1 intrathecal spinally per rat (n = 4);
O Rb1 50 μg: A group in which 50 μg of ginsenoside Rb1 was administered intranasally (n = 4) per rat;
FIG. 3C is one hour and two hours after administration of the ginseng-derived saponin extract according to the present invention to the animal model in which chronic pain occurs after 4 weeks of preparation of a central neuropathic pain animal model. And a graph showing the results of a heat hyperalgesia test at 4 hours;
Δ Vehicle: A group in which 10 μl of saline was injected into the spinal cavity per rat (n = 4);
◇ 20 μg of total saponin: group administered with 20 μg of ginseng-derived saponin extract per rat intranasally (n = 4);
□ 50 μg of total saponin: group administered with 50 μg of ginseng-derived saponin extract intraratically into the spinal cord (n = 4);
? Total saponin 100 μg: group administered 100 g of ginseng-derived saponin extract per rat intrathecalally (n = 4);
O Total saponin 200 μg: Group administered with 200 μg of ginseng-derived saponin extract per rat intrathecal (n = 4);
FIG. 3D shows that the CNS-derived saponin extract of the ginseng-derived saponin extract according to the present invention was administered in the spinal cord for 1 hour and 2 hours after the preparation of the central neuropathic pain animal model. And a graph showing the results of a heat hyperalgesia test at 4 hours;
◇ Vehicle: negative control group (n = 4) administered 10 μl of saline per spinal cord per rat;
10 μg of Rb1: group in which 10 μg of ginsenoside Rb1 was administered intranasally (n = 3) per rat;
B 25 μg of Rb1: group administered with 25 μg of ginsenoside Rb1 intrathecal spinally per rat (n = 4);
O Rb1 50 μg: A group in which 50 μg of ginsenoside Rb1 was administered intranasally (n = 4) per rat.
4 is a graph showing the inhibitory effect of central neuropathic pain reduction of the estrogen receptor:
FIG. 4A shows that the ginseng-derived saponin extract and the estrogen antagonist were administered to the animal model in which chronic pain occurred 4 weeks after the preparation of the central neuropathic pain animal model, respectively, or in the spinal cord for 1 hour, 2 hours. Graph showing results of mechanical allodynia testing at time and 4 hours;
O Total saponin 200 μg: Group administered with 200 μg of ginseng-derived saponin extract per rat intranasally alone (n = 4);
Sa 200 μg of total saponin + 50 μg of ICI: group administered 200 g of ginseng-derived saponin extract per rat and 50 μg of estrogen antagonist ICI in the spinal cord (n = 4);
50 μg of ICI: group administered with 50 μg of estrogen antagonist alone per spinal cord alone (n = 4);
Vehicle: negative control group (n = 4) administered 10 μl of saline solution per rat intrathecal spine;
FIG. 4B shows that Rb1 and an estrogen antagonist are administered to the animal model in which chronic pain develops after 4 weeks from the preparation of a central neuropathic pain animal model, or in the spinal cord, respectively, for 1 hour, 2 hours, and A graph showing the results of a mechanical allodynia test at 4 hours;
O Rb1 50 μg: A group in which 50 g of ginseng-derived saponin extract Rb1 was administered intranasally alone (n = 4) per rat;
B 50 μg Rb1 + 50 μg ICI: Group administered 50 g of ginseng-derived saponin extract Rb1 and 50 μg of estrogen antagonist ICI in the spinal cord (n = 4);
50 μg of ICI: group administered with 50 μg of estrogen antagonist ICI alone per spinal cord alone (n = 4);
◇ Vehicle: negative control group (n = 4) administered 10 μl of saline solution per rat intranasally;
FIG. 4C shows ginseng-derived saponin extract and estrogen antagonist, respectively, or in the spinal cord for 4 hours after the preparation of the central neuropathic pain animal model, and chronic pain. A graph showing the results of heat hyperalgesia testing at hours and 4 hours;
O Total saponin 200 μg: Group administered with 200 μg of ginseng-derived saponin extract per rat intranasally alone (n = 4);
Sa 200 μg of total saponin + 50 μg of ICI: group administered 200 g of ginseng-derived saponin extract per rat and 50 μg of estrogen antagonist ICI in the spinal cord (n = 4);
50 μg of ICI: group administered with 50 μg of estrogen antagonist alone per spinal cord alone (n = 4);
◇ Vehicle: negative control group (n = 4) administered 10 μl of saline per spinal cord per rat;
4D shows that 4 weeks after the preparation of the central neuropathic pain animal model, the animals with chronic pain were administered with Rb1 and the estrogen antagonist, respectively, or together with the spinal cord, 1 hour, 2 hours. A graph showing the results of heat hyperalgesia testing at hours and 4 hours;
O Rb1 50 μg: A group in which 50 μg of ginsenoside Rb1 was administered intranasally alone (n = 4) per rat;
B 50 μg Rb1 + 50 μg ICI: 50 μg of ginsenoside Rb1 per rat and 50 μg of estrogen antagonist ICI in the spinal cord (n = 4);
50 μg of ICI: group administered with 50 μg of estrogen antagonist ICI alone per spinal cord alone (n = 4); And
◇ Vehicle: Negative control group (n = 4) administered 10 μl of saline solution per rat intranasally.
Figure 5 is a graph showing the effect of inhibiting pain caused by the inflammatory response of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract:
FIG. 5A is a graph showing pain behavior (licking or lifting feet) observed after administration of ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract 30 minutes before injecting formalin causing an inflammatory response. ;
Vehicle: negative control group (n = 8) administered 10 μl of saline solution per rat into the spinal cord;
Sa 200 μg of total saponin: group administered with 200 μg of ginseng-derived saponin extract per rat intracranial cavity (n = 4);
50 μg Compound K: Group administered with 50 μg of ginsenoside Compound K per rat in the spinal cord (n = 4);
◇ 50 μg of Rb1: group administered with 50 μg of ginsenoside Rb1 per rat in the spinal cavity (n = 3);
ΔRg3 50 μg: A group in which 50 μg of ginsenoside Rg3 per rat was administered intranasally (n = 3);
FIG. 5B shows the pain behaviors (licking or lifting feet) observed as soon as injecting ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract, 30 minutes after injecting formalin causing an inflammatory response in rats. The graphs were divided into time zones of 0 to 10 minutes and 10 to 60 minutes;
Early phase: between 0 and 10 minutes after formalin administration;
Late phase: between 10 and 60 minutes of inflammatory response;
1 Vehicle: negative control group (n = 8) administered 10 μl saline intranasally;
2 μg of total saponin: group administered with 200 μg / rat of ginseng-derived saponin extract in the spinal cord (n = 4);
3 μg of Compound K: group administered with Compound K, a metabolite of ginsenoside Rb1, at 50 μg / rat (n = 4);
4 Rb1 50 μg: group administered intranasally with ginsenoside Rb1 50 μg / rat (n = 4); And
5 μg of Rg3: group administered in the spinal cavity with 50 μg / rat of ginsenoside Rg3 (n = 3).
Figure 6 is a figure confirming the inhibitory effect of microglia cells activity of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract:
FIG. 6A shows microglial cells at lamina 1 and 2 (lamina I and II) of the lumbar spinal cord olfactory region after oral administration of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract. The activity inhibition effect of was confirmed by immunohistochemical staining method;
Vehicle: negative control group (n = 4) orally administered 1 ml of saline once a day;
Minocycline; Positive control group (n = 4) administered 22.5 mg / kg of minocycline twice per day intraperitoneally;
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 4);
Compound K: group orally administered 7 mg / kg of ginsenoside Compound K once daily (n = 4);
Total saponin: Group administered orally with 50 mg / kg ginseng-derived saponin extract once daily (n = 4);
Rg3: group orally administered 11.5 mg / kg of ginsenoside Rg3 once daily (n = 4);
FIG. 6B shows the activated micro nerves in lamina 1 and 2 (lamina I and II) of the lumbar spinal cord olfactory region after administration of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract. A graph showing the ratio of glial cells to inactivated microglial cells;
Vehicle: A negative control orally administered with 1 ml of saline solution, in the vehicle figure shown in FIG. 6A, the microglial cell activity (black on the bar graph) and the total number of 67 cells in the tissue part surrounded by the dotted line. Inactive (grey in bar graph) (n = 4);
Minocycline; As a positive control group administered with 22.5 mg / kg of minocycline, in the Minocycline diagram shown in FIG. 6A, the microglial cell activity (black) and inactivation (in the total number of 64 cells in the dotted tissue region) Gray of bar graph) (n = 4);
Rb1: A group administered with 11.5 mg / kg ginsenoside Rb1, and in the Rb1 diagram shown in FIG. 6A, the activity of microglial cells (black color of the histogram) out of 61 cell numbers of the tissue part enclosed by the dotted lines. ) And inactive (gray bars) (n = 4);
Compound K: A group administered with 7 mg / kg of ginsenoside Compound K, and in the Compound K diagram shown in FIG. 6A, the activity of microglial cells among the total number of 60 cells in the tissue part surrounded by the dotted line (rod) Black in the graph) and inactive (grey in the bar graph) (n = 4);
Total saponins: A group administered with 50 mg / kg ginseng-derived saponin extract (n = 4), and in the total saponins figure shown in FIG. Indicates active (black on bar graph) and inactive (grey on bar graph);
Rg3: A group administered with 11.5 mg / kg of ginsenoside Rg3, and in the Rg3 diagram shown in FIG. 6A, the activity of microglial cells among the total number of 66 cells in the tissue part enclosed by the dotted lines (black on the bar graph) ) And inactive (grey on the bar graph) (n = 4).
FIG. 7 illustrates the fineness of lamina 1 and 2 (lamina I and II) of the lumbar spinal cord olfactory site after administration of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract in FIG. In the figure showing the activation of glial cells using immunohistostaining, an enlarged area of tissue surrounded by dotted lines is shown.
Vehicle: negative control group (n = 4) orally administered 1 ml of saline once a day;
Minocycline; Positive control group (n = 4) administered 22.5 mg / kg of minocycline twice per day intraperitoneally;
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 4);
Compound K: group orally administered 7 mg / kg of ginsenoside Compound K once daily (n = 4);
Total saponin: Group administered orally with 50 mg / kg ginseng-derived saponin extract once daily (n = 4); And
Rg3: group orally administered 11.5 mg / kg of ginsenoside Rg3 once daily (n = 4).
Figure 8 is a result of Western blot separation of spinal cord spinal cord 4 of the central neuropathic pain animal model to determine the effect of ginsenoside Rb1 and g-saponin-derived saponin extract on the activity of p-p38MAPK:
8A is a diagram showing the result of Western blot for confirming the activity of p-p38MAPK after administration of ginsenoside Rb1 and ginseng-derived saponin extract to a central neuropathic pain animal model;
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Min: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total-sap: group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5);
8B is a graph showing the expression level of p-p38MAPK based on the expression level of the control group in the result of the Western blot;
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Minocycline: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total saponins: Group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5); And
*: P <0.05.
9 is a western blot of spinal cord spinal cord isolated from a central neuropathic pain animal model to examine the effects of ginsenoside Rb1 and the activity of phosphorylated extracellular signal-regulated kinases (pERK) of ginseng-derived saponin extracts. The result of doing this is:
FIG. 9A is a diagram showing the result of Western blot for confirming the activity of pERK after administration of ginsenoside Rb1 and ginseng-derived saponin extract to a central neuropathic pain animal model; FIG.
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Min: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total-sap: group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5);
9B is a graph showing the expression level of pERK based on the expression level of the control group in the results of the Western blot;
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Minocycline: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total saponins: Group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5); And
*: P <0.05.
Figure 10 is to determine the effect of ginsenoside Rb1, and ginseng-derived saponin extract on the activity of pJNK (phosphorylated c-Jun N-terminal kinases), isolated spinal cord spinal cord 4 of the central neuropathic pain animal model The result of performing the western blot is:
FIG. 10A is a diagram showing the result of Western blot performed to confirm the activity of pJNK after administration of ginsenoside Rb1 and ginseng-derived saponin extract to a central neuropathic pain animal model; FIG.
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Min: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total-sap: group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5);
10B is a graph showing the expression level of pJNK in the western blot, based on the expression level of the control group;
Veh: negative control group (n = 5) orally administered 1 ml of saline once a day;
Minocycline: group intraperitoneally administered 22.5 mg / kg of minocycline twice daily (n = 5);
Total saponins: Group administered orally with ginseng-derived saponin extract of 50 mg / kg once daily (n = 5);
Rb1: group orally administered 11.5 mg / kg of ginsenoside Rb1 once daily (n = 5); And
*: P <0.05.
FIG. 11 is a graph showing the inhibitory effect of peripheral neuropathic pain of ginsenoside Rb1 and ginseng derived saponin extract:
FIG. 11A shows that a peripheral neuropathic pain animal model was prepared. After two weeks, the animal model with chronic pain was administered with the ginseng-derived saponin extract according to the present invention into the spinal cord by concentration, and the results of the mechanical allodynia test at 14, 15 and 16 days;
Vehicle: negative control (n = 4) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 4) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4);
11B shows that after two weeks have elapsed since the preparation of a peripheral neuropathic pain animal model, after administration of the ginseng-derived saponin extract according to the present invention into the spinal cord in an animal model having chronic pain, 14, A graph showing the results of cold allodynia testing at day 15 and 16;
Vehicle: negative control (n = 4) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 4) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4);
11C shows that after two weeks after the preparation of the peripheral neuropathic pain animal model, the animal model in which chronic pain occurs, after administration of ginseng-derived saponin extract according to the present invention by concentration, into the spinal cord, 14, A graph showing the results of a warm allodynia test from day 15 and 16;
Vehicle: negative control (n = 4) administered 1 ml saline;
∇ Minocycline; Positive control group (n = 4) administered 22.5 mg / kg minocycline;
Rb1: group receiving 11.5 mg / kg ginsenoside Rb1 (n = 4);
Compound K: group administered with a metabolite of 7 mg / kg ginsenoside Rb1 (n = 4);
Δ Total saponin: group administered with 50 mg / kg ginseng-derived saponin extract (n = 4);

Rg3: group receiving 11.5 mg / kg ginsenoside Rg3 (n = 4).

Hereinafter, the present invention will be described in detail.

The present invention ginseng ( Panax It provides a composition for the prevention and treatment of neuropathic pain containing ginseng ) derived extract or saponin isolated therefrom as an active ingredient.

In addition, the present invention provides a composition for preventing and treating neuropathic pain, which contains the compound represented by the following [Formula 1] as an active ingredient, isolated from ginseng-derived saponin extract:

[Formula 1]

;

;

Wherein, R1 is Glc _{2 -} is Glc or H;

R2 is Glc ₆ -Glc or H or Glc.

The neuropathic pain is preferably induced by peripheral nerve damage or central nerve damage, multiple sclerosis, including peripheral nervous system damage or spinal nerve injury, trigeminal neuralgia, diabetic neuropathy (diabetes neuropathy) ), Phantom limb pain, HIV infection, cancer, alcoholism, pain following chemotherapy, cancer treatment, atypical facial pain, post herpetic neuralgia, and neurological It is preferred to include, but is not limited to, neuropathic pain resulting from the disorder. In addition, the composition containing the ginseng-derived saponin extract as an active ingredient is preferred to reduce inflammatory pain, but is not limited thereto.

The composition preferably inhibits pain through the estrogen receptor, but is not limited thereto. In addition, the composition is preferably to inhibit the activity of the microglia cells in the spinal cord after spinal nerve trauma, it is preferable to inhibit the activity or expression of p-p38, pERK, and pJNK in the spinal cord, but is not limited thereto.

In the compound represented by the separation from the saponins derived from ginseng extract, Formula 1, to the compound represented by the [formula 2], Jean R1 of ginsenoside Rb1 is Glc _{2 -} Glc and R2 are those having from ₆ Glc -Glc desirable:

Wherein, R1 is Glc _{2 -} Glc and;

R2 is Glc ₆ -Glc.

In the compound represented by Formula 1, isolated from the ginseng-derived saponin extract, R 1 of ginsenoside Rg 3, which is a compound represented by Formula 3, is preferably Glc _2- Glc and R ₂ has H:

Wherein, R1 is Glc _{2 -} Glc and;

R2 is H.

In the compound represented by Formula 1, isolated from the ginseng-derived saponin extract, R 1 of ginsenoside Compound K, which is a compound represented by Formula 4, is preferably H, and R 2 has Glc:

Wherein R 1 is H;

R2 is Glc.

The ginseng-derived saponin extract is preferably prepared in the following steps, but is not limited thereto:

1) Dry Ginseng ( Panax ginseng ) extracting by adding an extraction solvent;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) under reduced pressure.

In the above method, the ginseng of step 1) can be used without limitation, such as those grown or commercially available.

The extraction solvent is preferably a solvent selected from water, an alcohol or a mixture thereof, preferably a lower alcohol of C ₁ to C ₂ or a mixed solvent thereof, more preferably using an aqueous 70% ethanol solution, but It is not limited. The amount of the extraction solvent is 2 to 15 times the dry ginseng weight. The extraction method may be an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, ultrasonic extraction and cold needle extraction, it may be preferably a cold needle extraction method. The temperature at the time of extraction is preferably 10 ° C. to 100 ° C., more preferably room temperature. The extraction time is 30 minutes to 4 hours, preferably 1 to 2 hours.

In the above method, the reduced pressure concentration in step 3) is preferably a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably lyophilized, but is not limited thereto.

In a specific embodiment of the present invention, to determine the neuropathic pain inhibitory effect of ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract, after preparing a central neuropathic pain animal model, according to the present invention Ginsenosides Rb1 and Rg3, Compound K, or ginseng derived saponin extract were administered. As a result, there was no change in exercise function by administration, but the pain inhibitory effect on mechanical allodynia and hyperalgesia was observed, and these effects were derived from ginsenosides Rb1 and Rg3, Compound K, or ginseng derived from the present invention. Observed in both saponin extracts (see FIG. 2).

In addition, in a specific embodiment of the present invention, 20, 50, 100, and 200 ㎍ to determine the pain inhibitory effect according to the administration concentration of ginseng-derived saponin extract when administered to a central neuropathic pain animal model, 20 ㎍ And 50 ㎍ concentration showed no effect at all and showed a concentration-dependent analgesic effect at 100 ㎍ and 200 ㎍ concentration (see Figure 3).

In addition, in a specific embodiment of the present invention, in order to confirm the specific mechanism of the pain inhibitory effect of ginsenoside Rb1 and ginseng-derived saponin extract according to the present invention, ginsenoside Rb1 in the central neuropathic pain animal model Or ginseng-derived saponin extract. As a result, when the estrogen antagonist was administered together, it was confirmed that the pain inhibitory effect observed when the Rb1 or ginseng-derived saponin extract was administered, and through these results, the ginsenoside Rb1 of the present invention and the saponin extract of ginseng It was confirmed that the analgesic effect is mediated through the estrogen receptor (see FIG. 4).

In addition, in a specific embodiment of the present invention, ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract of the present invention, in pain due to inflammation as well as pain caused by damage to the central and peripheral nerves The pain inhibitory effect of was observed. As a result, after administration of formalin subcutaneously, it was confirmed that between 10 minutes and 60 minutes when the inflammatory response began to appear, the inflammatory response was significantly reduced compared to the control group (see FIG. 5).

In addition, in a specific embodiment of the present invention, after administration of ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract in the central neuropathic pain animal model, the activation of microglial cells was observed. Using OX-42, a microglial cell marker, immunostaining was observed for lamina (lamina) I and II sites of the spinal cord olfactory region. Ginsenosides Rb1 and Rg3, Compound K, or ginseng derived from the present invention were observed. In the group administered with saponin extract, the number of activated microglial cells was confirmed to decrease. These results show that ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts according to the present invention inhibit pain by inhibiting activation of microglial cells (see FIGS. 6 and 7).

In addition, in a specific embodiment of the present invention, the activity of p-p38MAPK, pERK, and pJNK by administration of ginsenoside Rb1 and ginseng-derived saponin extract in the central neuropathic pain animal model was examined. Activation of p-p38MAPK, pERK, and pJNK is known to be closely related to microglial cell activation. Based on these findings, Western blot was performed by separating spinal cord 4 from the central neuropathic pain animal model. As shown in Figures 8 to 10, the expression of p-p38MAPK, pERK, and pJNK was significantly reduced in the ginseng-derived saponin extract and Rb1 of the present invention compared to the control group. In addition, this expression was observed to be more effectively reduced compared to minocycline (minocycline) used as a positive control (see FIGS. 8 to 10).

In addition, using the peripheral neuropathic pain model prepared by removing the peripheral nerve in the present invention, as a result of confirming the pain inhibitory effect of ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention, Similar to neuropathic animal models, pain inhibitory effects on mechanical allodynia, heat and cold hyperalgesia were observed, and these effects were found in both ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts according to the present invention. Was observed (see FIG. 11).

Therefore, the ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts according to the present invention have an inhibitory effect on neuropathic pain and inflammation-induced pain that each component can cause after spinal cord injury. , Ginsenoside Rb1 and Rg3, Compound K, or a composition containing one or more components selected from the group consisting of ginseng-derived saponin extract as an active ingredient can be usefully used as a composition for the prevention and treatment of neuropathic pain.

The composition for preventing and treating spinal cord injury of the present invention preferably includes 0.1 to 50% by weight of ginseng-derived saponin extract based on the total weight of the composition, but is not limited thereto. The composition containing ginseng derived saponin extract may further comprise suitable carriers, excipients and diluents commonly used in the preparation of the composition. Pharmaceutical dosage forms of the compositions may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The composition may be formulated in the form of powders, granules, tablets, capsules, soft capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate and sucrose in the composition. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the composition may be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be envisaged, eg, by intrathecal, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a health food for preventing and improving neuropathic pain containing ginseng ( Panax ginseng ) -derived saponin extract or saponin isolated therefrom as an active ingredient.

In addition, the present invention provides a health food for preventing and improving neuropathic pain containing the compound represented by the following [Formula 1] as an active ingredient, isolated from ginseng-derived saponin extract:

[Formula 1]

;

;

Wherein, R1 is Glc _{2 -} is Glc or H;

R2 is Glc ₆ -Glc or H or Glc.

In the compound represented by the separation from the saponins derived from ginseng extract, Formula 1, to the compound represented by the [formula 2], Jean R1 of ginsenoside Rb1 is Glc _{2 -} Glc and R2 are those having from ₆ Glc -Glc desirable:

(2)

;

;

Wherein, R1 is Glc _{2 -} Glc and;

R2 is Glc ₆ -Glc.

In the compound represented by Formula 1, isolated from the ginseng-derived saponin extract, R 1 of ginsenoside Rg 3, which is a compound represented by Formula 3, is preferably Glc _2- Glc and R ₂ has H:

(3)

;

;

Wherein, R1 is Glc _{2 -} Glc and;

R2 is H.

In the compound represented by Formula 1, isolated from the ginseng-derived saponin extract, R 1 of ginsenoside Compound K, which is a compound represented by Formula 4, is preferably H, and R 2 has Glc:

[Formula 4]

;

;

Wherein R 1 is H;

R2 is Glc.

The neuropathic pain is preferably induced by peripheral nerve damage or central nerve damage, multiple sclerosis, including peripheral nervous system damage or spinal nerve injury, trigeminal neuralgia, diabetic neuropathy (diabetes neuropathy) ), Phantom limb pain, HIV infection, cancer, alcoholism, pain following chemotherapy, cancer treatment, atypical facial pain, post herpetic neuralgia, and neurological It is preferred to include, but is not limited to, neuropathic pain resulting from the disorder. In addition, the composition containing the ginseng-derived saponin extract as an active ingredient is preferred to reduce inflammatory pain, but is not limited thereto.

The composition preferably inhibits pain through the estrogen receptor, but is not limited thereto. In addition, the composition is preferably to inhibit the activity of the microglia cells in the spinal cord after spinal nerve trauma, it is preferable to inhibit the activity or expression of p-p38, pERK, and pJNK in the spinal cord, but is not limited thereto.

Through the embodiments of the present invention, the composition containing the ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract, respectively, according to the present invention is caused by inflammation as well as pain caused by central nerve damage and peripheral nerve damage. An effect of suppressing the pain caused was observed, and it was confirmed that this pain-reducing effect is mediated by the estrogen receptor and is also shown by decreasing the expression of p-p38MAPK, pERK, and pJNK associated with inflammation.

Therefore, ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention a composition containing one or more ingredients selected from the group consisting of active ingredients for the prevention and improvement of neuropathic pain This can be usefully used.

There is no particular limitation on the kind of the food. Examples of foods to which the above-mentioned substances may be added include dairy products including drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, Beverages, alcoholic beverages, vitamin complexes, and the like, and include all healthy foods in the conventional sense.

The ginseng-derived saponin extract of the present invention may be added to a food as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the extract in the health food can be added at 0.01 to 15% by weight of the total food weight, the health beverage composition can be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the ginseng-derived saponin extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the ginseng-derived saponin extract of the present invention may contain a pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the ginseng-derived saponin extract of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

< Example  1> Ginseng  Preparation of Saponin Extract

Ginseng-derived saponin extract was extracted by cold needle at room temperature with 1 kg of 6 year old ginseng (Chungnam Geumsan) at 70% ethanol, and the freeze-dried powder was dissolved in water or buffer solution.

< Example  2> Ginseng  From saponin extract Gin Senocide Rb1  And Rg3 , Compound K Components

Cut 1 kg of 6-year-old ginseng (Gumsan, Chungnam) into a suitable size (1 ~ 2㎝), degreased with 10 liters of hexane (Hexane), extract the residue 5 times with 10 liters of methanol (MeOH), and concentrate it under reduced pressure. g was obtained. This was dissolved in 500 ml of water, extracted and concentrated four times with 3 L of butanol (BuOH) to obtain 85 g of ginseng butanol fraction (ginseng total fraction). The fractions were suspended in 8.5 L of water and 200 g (wet weight) of each of Bacteroides stercoris HJ-15, previously incubated in brain heart infusion mediu (Difco co., U.S.A.), and incubated at 37 ° C for 5 days. Then, extracted with 20 L of butanol four times and concentrated to obtain 45 g of processed ginseng fraction.

This was silica gel column chromatography [column size: 5 × 80 cm, developing solvent: CH ₂ Cl ₂ : methanol (20: 1 → 20: 2 → 20: 3: → 20: 4 → 20: 5 → 20: 6, each 1 L)]], fractions 1-6, and fractions 3 (3.1 g) and fractions 6 (10.3 g) were respectively reconstructed into silica gel columns [column size: 3 × 50 cm, developing solvent CHCl ₃ : Methanol: Compound K [20-0-β-D-glucopyranosyl-20 (S) -protopanaxadiol, 20-0-β-D- using H ₂ O (65:35:10, lower layer) 2 L] Glucopyranosyl-20 (S) -protopanaxadiol] (120 mg) and ginsenoside Rb1 (1.2 g) were isolated.

Ginsenoside Rb1 White needle , mp 197-198 ^o C ( dec .) FAB - MS (m / z) 1110 [M + 1] ⁺

Ginsenoside Rg3 White needle , mp 248-250 ^o C ( dec .) FAB - MS (m / z) 786 [M + 1] ⁺

Compound  K White needle , m.p. 219-221 ° C, FAB - MS  m / z: 623 [M + l] ⁺

2 g of ginseng dry matter was taken and extracted three times with 100 ml of methanol. The extract was concentrated under reduced pressure, and then 100 ml of water was added thereto and suspended. The extract was extracted three times with 100 ml of ether and concentrated under reduced pressure. Then, the resultant was extracted three times with 100 ml of butanol and concentrated under reduced pressure, and the obtained concentrate was dissolved in 100 ml of methanol to obtain 100 mg of saponin fraction. The content of each ginsenoside was determined by HPLC [YoungLin HPLC system: Kalam, Lichrosorb NH _{2 (} 25). X 0.4 cm, 5 mm, Merck Co., USA); Elution solvent, mixed solvent of solvent A (acetonitrile / water / isopropanol = 80: 5: 15) and solvent B (acetonitrile / water / isopropanol = 80: 20: 15), solvent A and solvent B solvent gradient: 0-20 min, solvent A: 0: 100 and 20-60 min at solvent B = 70: 30, solvent A: solvent B = 0: 100; Detector, ELSD800 (Alltech Asociates Inc., Deerfield, IL, USA).

The types and contents of total saponins contained in ginseng are shown in Tables 1 and 2 below. Rb1 is contained in ginseng and red ginseng, Rg3 is contained in red ginseng, and Compound K is a metabolite that is absorbed and converted into ginseng or red ginseng.

Saponin content of ginseng and saponin content according to temperature when making ginseng into red ginseng (w / w,%)           Ginseng               Red ginseng   100 ° c   110 ° c   120 ° c Rb1 0.56 ± 0.03 0.50 0.03 0.30 0.03 0.12 + 0.03 Rb2 0.52 ± 0.03 0.44 + 0.04 0.26 ± 0.04 0.10 ± 0.03 Rc 0.65 ± 0.03 0.57 ± 0.05 0.40 ± 0.08 0.17 ± 0.06 Rd 0.28 + 0.04 0.27 ± 0.03 0.20 + 0.04 0.14 ± 0.08 Re 0.38 ± 0.03 0.30 + 0.04 0.08 0.02 0.02 ± 0.01 Rf 0.11 + 0.02 0.12 + 0.03 0.10 ± 0.04 0.10 ± 0.04 Rb1 0.39 + 0.02 0.35 + 0.02 0.27 + 0.04 0.22 ± 0.04 Rg2 0.13 + - 0.01 0.20 + 0.02 0.32 ± 0.05 0.30 0.03 Rg3 n.d. ＊ 0.24 + 0.03 0.62 ± 0.06 1.32 ± 0.14 Rg5 n.d. ＊ 0.15 ± 0.03 0.35 ± 0.05 0.64 ± 0.08

Composition ratio of total (major) saponins in ginseng Types of Saponins Composition ratio (%) ginsenoside Ra 3.3 ginsenoside Rb1 22.9 ginsenoside Rb2 10.9 ginsenoside Rc 11.9 ginsenoside Rd 6.8 ginsenoside Rh2 0.1 ginsenoside Re 14.8 ginsenoside Rf 4.3 ginsenoside Rb1 18.6 ginsenoside Rg2 3.3 ginsenoside R0 2.3

The structures of ginsenosides Rb1, Rg3 and Compound K were reported as follows (Bae et al., 2010, J Neurochem).

[Formula 1]

;

;

Wherein, R1 is Glc _{2 -} is Glc or H;

R2 is Glc ₆ -Glc or H or Glc.

< Example  3> Animal Breeding Conditions

Seven-week-old adult male rats (weight 220-250 g) (Samgo, Osan, Gyeonggi-do) were used. The animals were housed in a 12-hour cycle in light and dark cycle rooms and were given free water and food. The following experiment was conducted according to the guidelines of animal experiments at Kyung Hee University.

< Example  4> backbone Neuropathic  Fabrication of Pain Animal Models

Spinal cord T8-T10 sections were exposed after anesthesia with chloral hydrate (500 mg / kg). As a spinal cord injury animal model, a 10g weight was dropped from a certain height to give trauma similar to human spinal cord injury, and NYU impactor was designed to quantify the trauma intensity on a computer. The rats were fixed in a clamp of the NYU impactor and then free-dropped a 10 g weight at 25 mm height to damage the spinal cord T9 site. The wound site was closed after confirming that a certain damage was applied within a predetermined error range through data on the damage intensity displayed on the computer. After the wound was disinfected with povidone solution, two dogs were raised in a cage, and artificially massaged the bladder three times a day to help urination.

< Example  5> peripheral Neuropathic  Production of Pain Animal Models

Rats (Sprague-Dawley rats, 150-200 g) were induced anesthesia with a gas mixture of 4% isoflurane and 96% oxygen, followed by 2-3% isopululan and 96% throughout the preoperative procedure. Anesthesia was maintained with an oxygen gas mixture. In order to cause neuropathic pain in the tail, the upper and inferior caudal trunks distributed in the tail of the rat are exposed, and 1 to 2 mm between the first and second shallow nerves as shown in FIG. Cut out (Na et al., 1994) (FIG. 1).

< Experimental Example  1> Gin Senocide Rb1  And Rg3 , Compound  K, and the center of ginseng-derived saponin extract Neuropathic  Pain inhibitory effect

After applying a trauma similar to the spinal cord injury in <Example 4>, using the prepared neuropathic pain animal model, the induction of neuropathic pain and ginsenosides Rb1 and Rg3, Compound K of the present invention, and Behavioral test was conducted to confirm the inhibitory effect of ginseng-derived saponin extract on central neuropathic pain. Specifically, the BBB locomoter test and pain response induced after injury were observed up to about 28 days after spinal cord injury. The pain behavior test was performed on 28, 29, and 30 days, and the mean value for 3 days was regarded as pre. Rats in which neuropathic pain was completely induced were selected and subjected to a pain behavioral test at 31, 32, and 33 days after spinal cord injury.

Ginseng-derived saponin extract (50 mg / kg, n = 4) and a single compound Rb1 isolated therefrom to a central neuropathic pain animal model having neuropathic pain after spinal cord injury according to the method of <Example 4> Neuropathic pain inhibitory effects were measured by oral administration of 11.5 mg / kg, n = 4, Rg3 (11.5 mg / kg, n = 4) and Compound K (7 mg / kg, n = 4), respectively. The negative control group (vehicle, n = 6) was orally administered with 1 ml of saline containing no ginseng saponin extract, and the positive control group was minocycline (22.5 mg / kg twice a day, intraperitoneal injection, n = 6). 6) was used.

Pain was confirmed before administration of ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin extract, and all behavioral tests after drug administration were conducted in blind study by a researcher who did not know whether the drug was administered.

<1-1> BBB  Exercise function test

The test is an experimental method that can be used to evaluate the recovery of various nerve bundles that control motor function.The score is divided from 0 to 21, and the score is given when the hind legs are not used at all and the score is 21 for normal rats. Higher indicates that motor function improved. Pre-adaptation training for the experimental environment by placing the rat in a plastic open field box 90 cm wide x 90 cm wide x 7 cm high for 4 minutes per animal, 3 days before the spinal cord trauma, once per day. Was carried out.

After administration of the spinal cord trauma, the ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention were administered for 3 consecutive days from the 31st day, and then subjected to a test to exclude individual biases in animal experiments. The experimenters did not know the information about each rat and scored 4 minutes of observation for each rat.

As a result, as shown in Figure 2A, the central neuropathic pain animal model according to the present invention ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract for 3 days each (after spinal cord injury, 31 Days, 32 days, and 33 days), the BBB total number of the motor function was 9.2 ± 0.7, the average value of all groups did not differ between groups (A in Fig. 2).

<1-2> mechanical Of mechanical allodynia  Measure

The present inventors attempted to measure the change in pain in response to mechanical stimulation to determine the effect of reducing or alleviating the pain behavior of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts.

The test animals were placed under a transparent plastic box (10 cm × 10 cm) on a wire mesh (3 mm × 3 mm). Adaptation was allowed for at least 20 minutes before the start of the sensory test. Von-Frey filaments were applied to both soles. Tactile (mechanical) sensitivity is logarithmic series of stiff nylon Bonfrey single filaments (3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18 mN, Stoelting, WoodDale, IL, USA; equivalent in grams to 0.4, 0.6 , 1.0, 2.0, 4.0, 6.0, 8.0 and 15.0) were used. The avoidance response of the foot to increased mechanical stimuli was assessed by applying sufficient force. The Bonfrey filament was applied to each foot for 3-4 seconds to bend completely. The 50% threshold was confirmed using the up-down method (Chaplan et al. 1994).

In summary, 2.0 g (4.31 mN) of filaments, the middle of eight bone filaments of 0.4 to 15 g, were used for the first time, beginning with the identification of the most sensitive areas at various sites. If the avoidance reaction of the foot did not occur, the filament of the next thickness was repeated. If there is a reaction, apply the weaker filament to the sensitive area. Stimulation was performed at 2 second intervals.

As shown in FIG. 2B, after 4 weeks of preparing the central neuropathic animal model according to the present invention, ginsenoside Rb1, Rg3, and Compound K, or ginseng-derived saponin extract according to the present invention. When not administered, the feet were lifted at 2.9 ± 0.1 g when stimulated with the soles of animal models with weighted and elastic filaments. This means that neuropathic pain was remarkably induced in the central neuropathic animal model prepared in the present invention.

After confirming that pain is induced in the central neuropathic animal model of the present invention, the analgesic effect of ginsenoside and ginseng-derived saponin extract according to the present invention was confirmed. As a result, as shown in FIG. 2B, the ginseng-derived saponin extract showed analgesic effect when administered repeatedly for 3 days, while Rb1 and Compound K showed analgesic effect gradually as treatment was repeated. On the third day, the last day of administration, the best effect was obtained. Rg3 began to suppress neuropathic pain from day 3 of oral administration (FIG. 2B). A positive control, minocycline, showed a significant analgesic effect by treating 22.5 mg twice a day and reducing pain response to mechanical stimulation for three days.

<1-3> columns Of heat hyperalgesia  Measure

The present inventors measured pain change with respect to temperature stimulation to determine whether the pain behavior of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract was reduced or alleviated.

Thermal susceptibility was assessed using the plantar inspection device as described by Hargreaves et al. (1988). Central neuropathic pain animal models on glass plates were acclimated for at least 20 minutes in an acrylic box. Under the glass plate, a device with a laser beam that can be moved to fit the surface of the animal's paws. The heat intensity of the light was adjusted by obtaining an average foot avoidance latency of about 15 seconds and the time limit set to 20 seconds to prevent tissue damage. Three thermal stimuli were given to both feet at 5-10 minute intervals. The average of the foot avoidance incubation times of each foot attempted three times indicated the degree of foot avoidance.

In the central neuropathic pain animal model according to the present invention, the pain-induced hypersensitivity test was performed. As shown in FIG. 2C, 4 weeks after the preparation of the central neuropathic animal model according to the present invention was performed. After lapse, when the ginsenoside Rb1, Rg3, and Compound K, or ginseng-derived saponin extract according to the present invention was not administered, the soles of the central neuropathic pain animal model were irritated with heat beams for 6.4 seconds. Confirmed that the wig was raised. This means that neuropathic pain was remarkably induced in the central neuropathic animal model prepared in the present invention.

After confirming that pain is induced in the central neuropathic animal model of the present invention, the analgesic effect of ginsenoside and ginseng-derived saponin extract according to the present invention was confirmed. As shown in FIG. 2C, the ginseng-derived saponin extract showed analgesic effect when administered repeatedly for 3 days, while Rb1 and Compound K showed analgesic effect gradually as treatment was repeated. The best effect was seen on day 3 of the day. Rg3 began to suppress neuropathic pain from day 3 of oral administration (FIG. 2C).

A positive control, minocycline, had a significant analgesic effect by treating twice a day and reducing pain response to thermal stimulation for three days.

In addition, as shown in FIG. 2D, when the ginseng-derived saponin extract and the single compound according to the present invention were administered, respectively, BBB values, mechanical allodynia, and heat hyperalgesia were measured. For BBB levels, no differences were found in all experimental groups (negative and positive controls, ginseng-derived saponin extracts, Rb1, Rg3, and Compound K), but for mechanical allodynia, Ginsenoside Rb1, and Compound K were administered. Similar to the minocycline used as a positive control group, the effect of significantly inhibiting the pain was observed compared to the control group. In case of heat hyperalgesia, the effect of significantly inhibiting pain was observed similarly to minocycline, in which Rb1 and Compound K were positive controls (FIG. 2D).

< Experimental Example  2> Ginseng  Depending on Saponin Extract Concentration Neuropathic  Confirmation of pain inhibitory effect

The present inventors, 20, 50, 100, 200 ㎍ the ginseng-derived saponin extract in the central neuropathic pain animal model prepared in Example 4 to determine the pain inhibitory effect according to the concentration of the ginseng-derived saponin extract After each administration intraperitoneally, the inhibitory effect of pain was observed for 4 hours.

As a result, as shown in FIG. 3A, when the ginseng-derived saponin extract 20, 50, 100, and 200 μg were administered into the spinal cord in the "pre" state 4 weeks after the preparation of the central neuropathic animal model, The analgesic effect on mechanical allodynia showed a concentration-dependent analgesic effect at concentrations of 100 μg and 200 μg (FIG. 3A).

In addition, as shown in B of FIG. 3, the heat hyperalgesia test showed the effect at 1 hour after administration only at a concentration of 200 μg (FIG. 3B).

In addition, when ginseng-derived saponin extract from 20 μg to 200 μg of high concentration was injected into the spinal cavity, the experimental animals exhibited no movement and no movement or sedation. Therefore, it was found that there was no effect of toxicity due to the high concentration of saponin extract.

< Experimental Example  3> The backbone of estrogen antagonists Neuropathic  Pain reduction inhibitory effect

After confirming the experimental results of the single injection of ginsenoside Rb1, Compound K and ginseng-derived saponin extract, the present inventors determined that the estrogen antagonist ICI 180782, a central neuropathic pain animal model, to determine whether their analgesic action is related to the estrogen receptor. Was administered once into the spinal cord.

First, after 4 weeks of preparing the central neuropathic pain animal model of <Example 4>, ginseng-derived saponin extract (200 μg / 10 μl) within the subarachnoid membrane between the lumbar vertebrae 5 to 6 The effect of single injection with Rb1 (50 μg / 10 μl), respectively, and co-administration with estrogen antagonist ICI (50 μg / 10 μl) and ginseng derived saponin extract or Rb1 Learned the relevance of

As shown in Figure 4, the result of testing the mechanical allodynia (mechanical allodynia), after administration of ginseng-derived saponin extract or ginsenoside Rb1 according to the present invention, respectively, when observed for 1 hour to 4 hours, administration 1 hour The most powerful analgesic effect was observed, which showed statistical significance (p <0.05). When the estrogen antagonist was administered with ginseng-derived saponin extract or ginsenoside Rb1, no analgesic effect was observed (A and B of FIG. 4).

In addition, as a result of examining the heat hyperalgesia, after administration of ginseng-derived saponin extract or ginsenoside Rb1 according to the present invention, respectively, for 1 to 4 hours, the most powerful analgesic after 1 hour of administration An effect was observed, which showed statistical significance (p <0.05). However, when the estrogen antagonist was administered with ginseng-derived saponin extract or Rb1, no analgesic effect was observed. Therefore, as a result of observing the pain response to the mechanical and thermal stimulation, it can be seen that ginseng-derived saponin extract and ginsenoside Rb1 mediate the pain effect through the estrogen receptor.

< Experimental Example  4> Gin Senocide Rb1  And Rg3 , Compound  K, or Ginseng  Inhibitory Effect of Saponin Extract on Inflammatory Pain

In order to measure the inflammatory pain inhibitory effect of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts, the present inventors injected adult male rats (weight 200-250) with 50 μl of 5% formalin solution subcutaneously. g) was used.

Specifically, the ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts of the present invention were injected 30 minutes prior to formalin injection, and then formalin solution was injected into the instep of adult male rats. The licking time or the lifting time was measured. Ginseng-derived saponin extracts contained 200 μg / rat (n = 4), Rb1 50 μg / rat (n = 3), Rg3 50 μg / rat (n = 3), Compound K 50 μg / rat (n = 4) Into the spinal cord. The control group was administered only saline without saponin extract (n = 8). After 30 minutes, as soon as the formalin solution was injected, the lifting duration and the licking duration were measured at 5 minute intervals for 1 hour after being placed in a transparent acrylic box.

In the untreated group, only formalin was injected subcutaneously without medication, and painful behavior was measured immediately after infusion. Pain behavioral response was measured in the same manner as in the measurement of mechanical allodynia and hyperalgesia described in <Example 1>.

As a result, as shown in Figure 5, between 0 ~ 10 minutes after the formalin administration (Early phase) was not significant between the groups, between 10 to 60 minutes (Late phase) when the inflammatory response (gatenoside Rb1 and Rg3 It was confirmed that the experimental group administered with saponin extract, Compound K, or ginseng derived from each group showed statistically significant (p <0.05) inflammatory pain inhibitory effect compared to the control group. Inflammation response time in the experimental group was reduced to less than 50% of ginseng-derived saponin extract, less than 7% of Rb1, less than 30% of Rg3, and less than 20% of Compound K. These results indicate that ginseng-derived saponin extracts and ginsenosides Rb1 and Rg3, Compound K isolated therefrom inhibit inflammatory pain as well as neuropathic pain (FIG. 5).

< Experimental Example  5> Gin Senocide Rb1  And Rg3 , Compound  K, or Ginseng  Micronerve of Saponin Extract Glial  Inhibitory effect

Ginseng-derived saponins in central neuropathic animal models according to the present invention Ginsenoside-derived saponin extract (50 mg / kg, n = 4) and ginsenosides isolated from the extract to determine the inhibitory effect of microglial cells on lamina I, II (lumbar 4) Rb1 (11.5 mg / kg, n = 4), Rg3 (11.5 mg / kg, n = 4), and Compound K (7 mg / kg, n = 4) were dissolved in tertiary distilled water, respectively, to the experimental animals 3 times a day. After oral administration for 1 day, 1 hour after oral administration on the last 3 days, tissues were obtained and subjected to immunohistochemical staining. The negative control group was orally administered 1 ml of saline once a day for 3 days (n = 4), and the positive control group was administered minocycline twice a day by intraperitoneal injection at a dose of 22.5 mg / kg for 3 days (n = 4).

Antibody-antibody reaction was performed with OX-42, a marker of microglial cells, to compare the number of activated microglial cells in the experimental and control groups.

Ginsenosides Rb1 and Rg3, Compound K, ginseng-derived saponin extract, vehicle, and minocycline according to the present invention one hour after the last day of drug administration, sodium pentobarbital (50 mg / kg) intraperitoneally After anesthesia by injection, 0.9% physiological saline solution was perfused through the left ventricle through the aorta to wash blood in the blood vessels, and then perfusion-fixed with 0.1 mol of phosphate buffer containing 4% paraformaldehyde. After spinal nerve 4 spinal nerves were removed, the tissues were postfixed in fixative solution for 2 hours, and then immersed in 30% sucrose solution at 4 ° C for 2-3 days until the tissues settle down. It was. The extracted tissue was cut into 10 mm using a frozen slicer, and then attached to a slide coated with gelatin and stored frozen until use.

Spinal cord slices cut to 10 mm thickness were subjected to immunohistochemical staining by ABC (Avidin-biotin-peroxidase complex) method using an antibody against OX-42, a microglial cell marker (Hsu et al., 1981). In order to increase the permeability of the tissue all reactions are carried out in 0.05 molar phosphate buffer (PBS-Tx) containing 0.3% Triton X-100. Tissue sections were reacted in PBS-Tx solution containing 10% normal goat serum (NGS) and 1% bovine serum albumin (BSA) for 1 and a half hours to prevent non-specific antigen-antibody reactions. , And reacted with OX-42, GFAP (Chemicon, USA) primary antibody diluted at 4 ° C for 24 hours (dilution determined during the experiment). The tissue sections were washed three times for 10 minutes with 0.05M PBS-Tx, and then immersed in a secondary antibody solution (biotinylated goat anti-mouse IgG) diluted 1: 200 and reacted at room temperature for 2 hours. Again washed three times with PBS-Tx for 10 minutes and reacted with ABC solution for 2 hours at room temperature. Wash three times with phosphate buffer three times for 10 minutes, and develop a color reaction at room temperature for 5 to 10 minutes (adjust the time while continuing to observe) in phosphate buffer containing 0.01% hydrogen peroxide and 0.05% DAB. After washing three times with phosphate buffer solution three times for 10 minutes, and then three times for 5 minutes in tertiary distilled water, dehydrated with ethyl alcohol and xylene in increasing order and put on a cover glass through a transparent process. Made into a sample.

The degree of expression of glial cells of the spinal cord olfactory cells was divided into the activated or restoring state of immunostained cells and counted and compared.

The expression level of OX-42 was analyzed by comparing the digital image of the spinal cord obtained through the microscope with the control software (Scion image, Scion Corp. USA).

As a result, as shown in FIG. 6A, the number of activated microglial cells was minocycline (minocycline) used as a positive control in a group administered with ginsenoside Rb1 and Rg3, Compound K, or ginseng derived saponin extract, respectively. It was confirmed that the decrease by the group administered. In particular, the group administered with Rb1, Compound K was confirmed that the number of activated microglial cells more significantly reduced than the group administered with minocycline (A of FIG. 6).

In addition, when counting the activated or inactivated microglial cells through the staining results, the number of activated microglial cells in the group administered with minocycline used as a positive control group decreased compared with the group administered with saline used as a negative control group. This trend was attributed to the administration of ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extracts, respectively. All were observed in the group. Among them, in particular, the group administered with ginsenoside Rb1 and Compound K showed a result of reducing the activity of microglial cells more effectively than minocycline (Fig. 6B). These results indicate that activated microglial cells in the spinal cord smell increase pain (Hains and Waxman, 2006 Detloff et al., 2008 Hulsebosch, 2008; Tan et al., 2009), which are isolated from ginseng-derived saponin extracts. Ginsenosides Rb1, Rg3 and Compound K means that inhibit the pain by preventing the activation of microglial cells (Fig. 6).

< Experimental Example  6> Gin Senocide Rb1  And Rg3 , Compound  K, or Ginseng  Of saponin extract p38MAPK , pERK , And pJNK  Effect on activity

The present inventors administered ginsenoside Rb1 and Rg3, Compound K, or ginseng-derived saponin extract to the central neuropathic pain animal model prepared in Example 4, To determine the effect on p - p38MAPK, pERK, and pJNK activity.

Specifically, ginsenoside Rb1 (11.5 mg / kg, n = 5) and ginseng-derived saponin extract (50 mg / kg, n = 5) were orally administered once a day for 3 days. 1 ml of saline was administered orally once a day for 3 days (n = 5), and the positive control group received minocycline intraperitoneally twice a day at 22.5 mg / kg (n = 5). After administration of ginsenoside Rb1, ginseng-derived saponin extract, physiological saline and minocycline for 3 days, 1 hour after 3 days of administration, lumbar spinal cord 4 was separated and western blotting was performed.

Specifically, lysis buffer [1% Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na ₂ P ₂ O ₇ , 10 mM NaF, 1 μg / Intracellular proteins were obtained using ml aprotinin, 10 μg / ml leupeptin, 1 mM vanadate, and 1 mM PMSF] and subjected to SDS-PAGE. After transfer to a nitrocellulose membrane, p-p38MAPK (1: 1,000, Cell signaling), pERK (1: 1,000, Cell Signaling), pJNK (1: 1,000, Cell Signaling) antibody Western blot was performed using the protocol shown in the data sheet for each antibody.

As a result, as shown in Figures 8 to 10, the expression levels of p-p38MAPK, pERK, and pJNK in the group administered ginseng-derived saponin extract and Rb1, respectively, was significantly reduced compared to the negative control, It was more remarkable than the administration of the positive control group minocycline. This is a result showing that the ginseng-derived saponin extract and Rb1 exhibits a pain inhibitory effect by inhibiting the expression amount of a factor related to pain (FIGS. 8 to 10).

< Experimental Example  7> Gin Senocide Rb1  And Rg3 , Compound  K, and Ginseng  Peripheral of saponin extract Neuropathic  Pain inhibitory effect

Using the peripheral neuropathic pain animal model prepared by damaging the tail nerve in Example 5, the cause of peripheral neuropathic pain and ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin of the present invention. Behavioral test was conducted to confirm the inhibitory effect of the extract on peripheral neuropathic pain. Specifically, the pain response induced about 14 days after peripheral nerve injury was observed. The pain behavior test was performed on 14, 15, and 16 days, and the average value over 3 days was regarded as pre-14d. Rats in which neuropathic pain was completely induced were selected and subjected to a pain behavioral test 14 days, 15 days, and 16 days after spinal cord injury.

Ginseng-derived saponin extract (50 mg / kg, n = 4) to a peripheral neuropathic pain animal model that developed neuropathic pain after peripheral nerve injury using the method of pain behavior test used in Experimental Example 1 above; Neuropathy by oral administration of single compound Rb1 (11.5 mg / kg, n = 4), Rg3 (11.5 mg / kg, n = 4) and Compound K (7 mg / kg, n = 4) The pain inhibitory effect was measured. The negative control group (vehicle, n = 4) was orally administered with 1 ml of physiological saline containing no ginseng derived saponin extract, and the positive control group was minocycline (22.5 mg / kg twice a day, intraperitoneal injection, n = 4). 6) was used.

Pain was confirmed before administration of ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin extract, and all behavioral tests after drug administration were conducted in blind study by a researcher who did not know whether the drug was administered.

<7-1> mechanical Of mechanical allodynia  Measure

Peripheral neuropathic pain animal models were placed in round acrylic barrels (5.5 × 15, 6.5 × 18 cm, used according to body size), pulled out of tails only, and placed on plates. Measurement of mechanical allodynia was 50% by stimulating the tail by Up & Down method (Chaplan et al, 1994) using various von Frey hair (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 15.0 g). The grams (g) of von Frey hair with avoidance reactions were calculated. It was determined that mechanical allodynia occurred as a statistically significant decrease in the avoidance response threshold compared to before nerve injury.

As a result, as shown in FIG. 11A, in the pre-OP condition, minocycline, a positive control group, was treated once a day (22.5 mg twice a day, intraperitoneally) and treated mechanically for three days. It showed a significant analgesic effect by reducing pain response to stimuli. In addition, after administration of ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin extracts according to the present invention, it was observed that pain was remarkably reduced similarly to minocycline used as a positive control (FIG. 11A). Specifically, ginseng-derived saponin extract showed analgesic effect when administered repeatedly for 3 days, while Rb1 and Compound K showed analgesic effect gradually as treatment was repeated, and the best on the third day of the last day of administration. Effect.

<7-2> cold Cold allodynia  Measure

Peripheral neuropathic pain animal model was placed in a round acrylic container, and the tail was taken out and drooped, and then placed in 4 ° C. water to measure the time until the tail showed an avoidance reaction. Experimental results were obtained by the mean of five measurements at intervals of 5 minutes. When not avoided by 15 seconds, the tail was pulled out of the water to prevent tail sensitization, resulting in 15 seconds. In cases of significant early avoidance than before surgery, it was estimated to be the result of cold allodynia.

As a result, as shown in FIG. 11B, in the pre-OP state, the positive control group minocycline was treated once a day (22.5 mg twice a day, intraperitoneal injection) in cold condition for 3 days as shown in FIG. It has shown significant analgesic effects by reducing pain response to allodynia. In addition, after administration of ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin extracts according to the present invention, similar to minocycline used as a positive control, it was observed to significantly reduce pain for cold allodynia (FIG. 11B). ). Specifically, ginseng-derived saponin extract showed analgesic effect when administered repeatedly for 3 days, while Rb1 and Compound K showed analgesic effect gradually as treatment was repeated, and the best on the third day of the last day of administration. Effect.

<7-3> on Of warm allodynia  Measure

The method for measuring all allodynia was carried out in the same manner as the cold allodynia of <7-2>, but was performed at a temperature of 40 ° C. The results of allodynia were presumed to be avoided significantly faster than before peripheral nerve damage.

As a result, as shown in FIG. 11C, in the pre-OP state, the positive control group minocycline was treated once a day (22.5 mg twice a day, intraperitoneal injection) for 3 days as shown in FIG. It has shown significant analgesic effects by reducing pain response to allodynia. In addition, after administration of ginsenosides Rb1 and Rg3, Compound K, and ginseng-derived saponin extracts according to the present invention, it was observed that the pain for allodynia remarkably remarkably similar to minocycline used as a positive control (FIG. 11C). ). Specifically, ginseng-derived saponin extract showed analgesic effect when administered repeatedly for 3 days, while Rb1 and Compound K showed analgesic effect gradually as treatment was repeated, and the best on the third day of the last day of administration. Effect.

The preparation examples for the compositions of the present invention are given below.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Sanje  Produce

2 g of saponin extract derived from ginseng

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

Ginsenoside Rb1 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

Compound K 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

Ginsenoside Rg3 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

Ginsenoside Rb1 150 mg

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

&Lt; Preparation Example 2 > Production of food

<2-1> Manufacture of confectionery products

0.5 to 5.0 parts by weight of ginseng-derived saponin extract of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

<2-2> Manufacture of dairy products

5 to 10 parts by weight of ginseng-derived saponin extract of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-3> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The ginseng-derived saponin extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the dry powder of the ginseng-derived saponin extract of the present invention were prepared by blending in the following ratio.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Dry powder of ginseng-derived saponin extract (3 parts by weight),

(0.5 parts by weight), and

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

Ginseng-derived saponin extract 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Add 900 ml of purified water

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the manufacture of health food of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

The composition containing ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention as an active ingredient may be substituted for drugs currently used clinically if used for neuropathic pain inhibition or treatment. In addition, it can improve the quality of life of the patient and reduce the economic and social loss in that it can compensate for the disadvantages such as side effects of taking a long time. Furthermore, pain after trigeminal neuralgia, diabetes neuropathy, phantom limb pain, multiple sclerosis, chemotherapy, HIV treatment, cancer, alcoholism, cancer treatment Tissue damage and the like based on the results additionally confirmed in the present invention, including neuropathic pain caused by various causes, including atypical facial pain, post herpetic neuralgia, neurological disorders, and the like. It can be expected that a good therapeutic effect can be expected even for the invasive pain caused by the disease. Therefore, if a composition comprising ginsenosides Rb1 and Rg3, Compound K, or ginseng-derived saponin extract according to the present invention as an active ingredient is developed as an agent for inhibiting and treating neuropathic pain, it can reduce the pain of patients suffering from chronic pain. In this regard, it can be used to develop pain treatment drugs.

Claims (2)

Health food for preventing and improving neuropathic pain containing ginsenoside Rg3 as an active ingredient, which is a compound represented by the following [Formula 3]:
(3)

;
Wherein, R1 is Glc _{2 -} Glc and;
R2 is H.
The method of claim 1, wherein the neuropathic pain is neuropathic pain prevention and improvement health foods, characterized in that induced by peripheral nerve damage or central nerve damage.

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