COMPOSITION COMPRISING THE EXTRACT OF HERBAL COMBINATION FOR PREVENTING OR TREATING DIABETIC PERIPHERAL NEUROPATHY

Abstract

Disclosed are a pharmaceutical composition and a health functional food for the prevention and treatment of diabetic peripheral neuropathy, comprising an herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica (w/w). Having the ability to synergistically increase in vivo levels of nerve growth factor, compared to the extracts from the herbs alone or herb mixtures of other weight ratios, the mixed herb extract is effective for preventing the apoptosis of nerve cells and promoting nerve regeneration. Thus, it can be applied to pharmaceutical compositions and health functional foods preventive and curative of diabetic peripheral neuropathy.

Claims

1. A method for treating a diabetic peripheral neuropathy in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an extract of a mixture comprising Dioscorea Rhizoma and a rhizome of Dioscorea nipponica.

2. The method of claim 1, wherein the diabetic peripheral neuropathy exhibits a symptom selected from the group consisting of a burning discomfort, a stingy ache, soreness, a straining feeling, loss of tactile sensation, vibratory sensation or temperature sensation, and a combination thereof.

3. (canceled)

4. The method of claim 1, wherein the Dioscorea Rhizoma is a rhizome of Dioscorea batatas Decaisne or Dioscorea japonica Thunberg.

5. The method of claim 1, wherein the rhizome of Dioscorea nipponica is a rhizome of Dioscorea nipponica Makino.

6. The method of claim 1, wherein the pharmaceutical composition comprises a suitable carrier, excipient or diluent selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

7. The method of claim 1, wherein the pharmaceutical composition is in a solid form.

8. The method of claim 1, wherein the pharmaceutical composition is in a liquid form.

9. The method of claim 4, wherein the Dioscorea Rhizoma is steamed and dried.

10. The method of claim 1, wherein the Dioscorea Rhizoma and the rhizome of Dioscorea nipponica are present in the mixture at a ratio of 3.5:1.

11. The method of claim 1, wherein the pharmaceutical composition comprises: (a) 100 mg of the extract; (b) 3.0 mg of sodium metabisulfite; (c) 0.8 mg of methylparaben; and (d) 0.1 mg of propylparaben; wherein the pharmaceutical composition is in an injectable form.

12. The method of claim 1, wherein the pharmaceutical composition comprises: (a) 100 mg of the extract; (b) 100 mg of lactose; (c) 100 mg of starch; and (d) a pharmaceutically acceptable amount of magnesium stearate; wherein the pharmaceutical composition is in a tablet form.

13. The method of claim 1, wherein the pharmaceutical composition comprises: (a) 100 mg of the extract; (b) 50 mg of lactose; (c) 50 mg of starch; (d) 2 mg of talc; and (e) a pharmaceutically acceptable amount of magnesium stearate; wherein the pharmaceutical composition is in a capsule form.

14. The method of claim 1, wherein the pharmaceutical composition comprises: (a) 100 mg of the extract; (b) 20 mg of sugar; (c) 20 mg of isomeroase; and (d) a pharmaceutically acceptable amount of lemon flavor; wherein the pharmaceutical composition is in a liquid form.

15. The method of claim 1, wherein the pharmaceutical composition is administered at a dose from 0.01 g/kg to 10 g/kg.

16. The method of claim 1, wherein the pharmaceutical composition is administered orally, intrarectally, intravenously, intramuscularly, hypodermically, endometrially, or intracerebroventicularly.

17. A pharmaceutical composition comprising an extract of a mixture comprising Dioscorea Rhizoma and a rhizome of Dioscorea nipponica, wherein the mixture is at a weight ratio of 3.5:1 (w/w) of the Dioscorea Rhizoma and the rhizome of Dioscorea nipponica.

18. The pharmaceutical composition of claim 17, which is capable of treating a diabetic peripheral neuropathy.

19. A health functional food comprising an extract of a mixture comprising Dioscorea Rhizoma and a rhizome of Dioscorea nipponica and a food additive, wherein the mixture is at a ratio of 3.5: 1 (w/w) of the Dioscorea Rhizoma and the rhizome of Dioscorea nipponica.

20. A method of preparing the pharmaceutical composition of claim 1 comprising: (a) providing a mixture comprising dried Dioscorea Rhizoma and a rhizome of Dioscorea nipponica; and (b) precipitating an extract from the mixture.

21. The method of claim 20, wherein the Dioscorea Rhizoma and the rhizome of Dioscorea nipponica (w/w) are present at a ratio of 3.5:1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 is of electron photographs showing the effect of the extract of Example 1 on the sciatic nerve of type 1 diabetes mice.

[0048] FIG. 2 is of electron photographs showing the effect of the extract of Example 1 on the sciatic nerve of type 1 diabetes rats.

MODE FOR THE INVENTION

[0049] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1

[0050] Preparation of Extract from Mixture of Dioscorea Rhizoma and Dioscorea Nipponica

[0051] Dioscorea Rhizoma and Dioscorea nipponica, both in a dry condition, were purchased from a herb medicine shop in Kyoungdong market, Korea. After impurities were removed therefrom, the herbs were chopped with a cutter and mixed at a weight ratio of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica. To 2 kg of the mixture was added 10 L of a 50% ethanol solution, followed by incubation at room temperature for 48 hours with stirring. The herb mixture was removed by filtration, and the filtrate was concentrated in a vacuum and freeze dried to afford a mixed herb extract (crude extract) (see Table 1).

TABLE-US-00001 TABLE 1 Yield of Inventive Combined Herbal Extract Crude Amount of Material Amount of Extract'n Time of Extract Yield Ex. D. Rhizoma D. nipponica Solvent Solvent Wash Temp. Extract'n (g) (%) Ex. 1 1.55 kg 0.45 kg 50% 10 L 1 L Room 2 days 212.85 10.64 EtOH Temp.

COMPARATIVE EXAMPLE 1

Preparation of Dioscorea Rhizoma Extract

[0052] A Dioscorea Rhizoma extract (crude extract) was prepared in the same manner as in Example 1, with the exception that 2 kg of Dioscorea Rhizoma was used instead of 2 kg of the mixture (see Table 2).

COMPARATIVE EXAMPLE 2

Preparation of Dioscorea Nipponica Extract

[0053] A Dioscorea nipponica extract (crude extract) was prepared in the same manner as in Example 1 with the exception that 2 kg of Dioscorea nipponica was used instead of 2 kg of the mixture (see Table 2).

TABLE-US-00002 TABLE 2 Yield of Respective Extracts from D Rhizoma and D. Nipponica Crude # of Amount of Material Amount Of Extract'n Time of Extract Yield C. Ex. D. Rhizoma D. nipponica Solvent Solvent Wash Temp. Extract'n (g) (%) 1 2 kg — 50% 10 L 1 L Room 2 days 253.8 12.69 EtOH Temp. 2 — 2 kg 50% 10 L 1 L Room 2 days 160.0 8.00 EtOH Temp.

COMPARATIVE EXAMPLE 3 TO 10

[0054] Preparation of Extracts from Mixtures of Dioscorea Rhizoma and Dioscorea Nipponica

[0055] The same herbs Dioscorea Rhizoma and Dioscorea nipponica as used in Example 1 were used. Dioscorea Rhizoma and Dioscorea nipponica were chopped with a cutter and mixed at the weight ratios listed in Table 3. To 2 kg of each of the mixtures was added 10 L of a 50% ethanol solution, followed by incubation at room temperature for 48 hours with stirring. The herb mixtures were removed by filtration, and the filtrate was concentrated in a vacuum and freeze dried to afford mixed herb extracts (crude extracts).

TABLE-US-00003 TABLE 3 Yield of Combined Extract of Mixture of D. Rhizoma and D. Nipponica Crude # of Amount of Material Solvent Extract'n Time of Extract Yield C. Ex. D. Rhizoma D. nipponica Kind Amount Washing Temp. Extract'n (g) (%) 3 1 1 50% 10 L 1 L Room 2 days 179.05 8.95 EtOH Temp. 4 1.33 0.67 50% 10 L 1 L Room 2 days 135.96 6.80 EtOH Temp. 5 1.67 0.33 50% 10 L 1 L Room 2 days 160.07 8.00 EtOH Temp. 6 1.82 0.18 50% 10 L 1 L Room 2 days 138.75 6.93 EtOH Temp. 7 0.67 1.33 50% 10 L 1 L Room 2 days 153.56 7.68 EtOH Temp. 8 0.45 1.55 50% 10 L 1 L Room 2 days 139.15 6.96 EtOH Temp. 9 0.33 1.67 50% 10 L 1 L Room 2 days 181.06 9.05 EtOH Temp. 10 0.18 1.82 50% 10 L 1 L Room 2 days 146.43 7.32 EtOH Temp.

TEST EXAMPLE 1

[0056] Test for In vivo Level of Nerve Growth Factor in Normal Mouse Model

[0057] Ability of the crude extract with 50% ethanol in Example 1 to induce the secretion of nerve growth factor in vivo was evaluated (O. Arrieta and J. Sotelo et al., Retinoic acid increases tissue and plasma contents of nerve growth factor and prevents neuropathy in diabetic mice. European Journal of Clinical Investigation (2005) 35).

[0058] Male ICR mice, each weighing 25-30 g, were acclimated for 1 week at 22-24° C. at a relative humidity of 60-80% while being fed with water and a standard diet. They were divided into groups according to weight. The extracts prepared in the above Examples were orally administered in predetermined amounts to the mice, with Gabapentin used as a positive control at a dose of 100 mg/kg, 16 hours after which blood was sampled from the mice. They were then sacrificed to obtain the sciatic nerve and the salivary glands.

[0059] After the blood samples were centrifuged at 10,000 rpm for 10 min, the levels of nerve growth factor in the plasma thus separated were measured by ELISA (ABS 450 nm). The sciatic nerve and the salivary glands were weighed and a solution containing 100 mM Tris-HCl, 1M NaCl, 2% BSA, 4 mM EDTA, 2.0% Trtion.circle-solid.X-100, 0.02% sodium azide. 0.1 mg/ml pepstatin A, 5 mg/ml aprotonin, 0.5 mg/ml antipain, 167 mg/ml benzamidine, and 5.2 mg/ml PMSF was added in amounts proportional to the weights, followed by homogenization. The homogenates were incubated for 15 min with 1N HCl in an amount of 1 μl/50 μl for oxidization and neutralized with 1N NaOH in an amount of 1 μl/50 μl Centrifugation was performed at 10,000 rpm for 10 min to obtain supernatants. Like plasma, the sciatic nerve and the salivary glands were quantitatively analyzed for the level of nerve growth factor.

[0060] Data are summarized in Table 4 for plasma NGF levels, in Table 5 for sciatic NGF levels, and in Table 6 for salivary gland NGF levels.

TABLE-US-00004 TABLE 4 NGF Level in Plasma Amount of Weight NGF material (kg) ratio of D. Level D. D. Rhizoma:D. Dose in Plasma Group Rhizoma nipponica nipponica (mg/kg) (pg/ml) control 601.5 Ex. 1 1.55 0.45 3.5:1.sup.  100 1106.9 C. Ex. 1 2 — — 100 606.2 C. Ex. 2 — 2 — 100 602.1 C. Ex. 3 1 1 1:1 100 663.6 C. Ex. 4 1.33 0.67 1:2 100 689.9 C. Ex. 5 1.67 0.33 5:1 100 725.6 C. Ex. 6 1.82 0.18 10:1  100 696.8 C. Ex. 7 0.67 1.33 1:2 100 607.0 C. Ex. 8 0.45 1.55 .sup. 1:3.5 100 656.0 C. Ex. 9 0.33 1.67 1:5 100 642.6 C. Ex. 10 0.18 1.82  1:10 100 640.7 Gabapentin 100 610.4

[0061] When administered at the same dosage, as can be seen in Table 4, the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, and the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10 were observed to induce the secretion of nerve growth factor in plasma to a degree similar to that of the control. In contrast, the extract of Example 1 increased the plasma NGF level over that obtained by the Dioscorea Rhizoma extract, the Dioscorea nipponica extract, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios.

[0062] Increasing the level of nerve growth factor in plasma, a path through which nerve growth factor moves toward targets, indicates promoting the secretion of nerve growth factor. Thus, the extract from a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica (w/w) in accordance with the present invention further promotes the secretion of nerve growth factor to significantly raise the level of NGF in vivo, compared to the extracts from Dioscorea Rhizoma alone, Dioscorea nipponica alone or from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios.

TABLE-US-00005 TABLE 5 NGF Level in Sciatic Nerve NGF Amount of Weight Level material(kg) ratio of D. in Sciatic D. D. Rhizoma:D. Dose Nerve Group Rhizoma nipponica nipponica (mg/kg) (pg/mg) control 3.3 Ex. 1 1.55 0.45 3.5:1.sup.  100 7.5 C. Ex. 1 2 — — 100 3.8 C. Ex. 2 — 2 — 100 3.6 C. Ex. 3 1 1 1:1 100 4.6 C. Ex. 4 1.33 0.67 1:2 100 4.1 C. Ex. 5 1.67 0.33 5:1 100 4.0 C. Ex. 6 1.82 0.18 10:1  100 3.2 C. Ex. 7 0.67 1.33 1:2 100 5.1 C. Ex. 8 0.45 1.55 .sup. 1:3.5 100 4.6 C. Ex. 9 0.33 1.67 1:5 100 4.0 C. Ex. 10 0.18 1.82  1:10 100 4.9 α-Lipoic 50 5.0 acid Gabapentin 100 4.2

[0063] Observations showed that, when administered at the same dosage, as can be seen in Table 5, the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, and the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10 secreted nerve growth factor in the sciatic nerve to a degree similar to that of the control. In contrast, the extract of Example 1 increased the sciatic nerve NGF level over that obtained by the Dioscorea Rhizoma extract, the Dioscorea nipponica extract, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios.

[0064] Accordingly, the extract from a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica in accordance with the present invention can induce the secretion of NGF into the blood, which results in a significant increase in the level of NGF in the sciatic nerve, the target tissue.

TABLE-US-00006 TABLE 6 NGF Level in Salivary Gland NGF Amount of Weight Level material(kg) ratio of D. in Salivary D. D. Rhizoma:D. Dose Gland Group Rhizoma nipponica nipponica (mg/kg) (ng/mg) control 166.1 Ex. 1 1.55 0.45 3.5:1.sup.  100 221.7 C. Ex. 1 2 — — 100 171.6 C. Ex. 2 — 2 — 100 170.1 C. Ex. 3 1 1 1:1 100 206.2 C. Ex. 4 1.33 0.67 2:1 100 193.3 C. Ex. 5 1.67 0.33 5:1 100 177.4 C. Ex. 6 1.82 0.18 10:1  100 202.5 C. Ex. 7 0.67 1.33 1:2 100 173.4 C. Ex. 8 0.45 1.55 .sup. 1:3.5 100 200.2 C. Ex. 9 0.33 1.67 1:5 100 181.2 C. Ex. 10 0.18 1.82  1:10 100 188.1 α-Lipoic 50 164.4 acid Gabapentin 100 193.1

[0065] As is apparent from the data of Table 6, when administered at the same dosage, the

[0066] Dioscorea Rhizoma extract of Comparative Example 1 and the Dioscorea nipponica extract of Comparative Example 2 increased the level of NGF in the salivary glands by 2{tilde over ( )}3% only, compared to the control while the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10 induced the secretion of NGF in the salivary glands to degrees similar to or less than those obtained by the positive control gabapentin. In contrast, the level of NGF in the salivary glands was 33.5% increased by the extract of Example 1. That is, the extract of the present invention allowed for a significant increase in the level of NGF in the salivary gland, compared to the extracts from Dioscorea Rhizoma alone, Dioscorea nipponica alone or mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios. Increasing the level of NGF in the salivary gland, which functions as a source of NGF, means promoting the synthesis of NGF.

[0067] Consequently, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica (w/w) can significantly raise the level of NGF in vivo.

[0068] Hence, thanks to its ability to significantly raise the level of NGF in vivo, the herb extract of a mixture of 3.5:1 DioscoreaRhizoma:Dioscorea nipponica (w/w) in accordance with the present invention can be applied to a pharmaceutical composition or a health functional food for the prevention and treatment of diabetic peripheral neuropathy.

TEST EXAMPLE 2

[0069] Test for Effects on Pain, NGF Secretion, and Neurodegeneration in SD Male Rats with Streptozotocin-Induced Type 1 Diabetes

[0070] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica, prepared in Example 1, and the extracts of Comparative Examples 1 to 10 were evaluated for effects on pain, in vivo NGF level and neurodegeneration in SD male rats with type 1 diabetes.

[0071] SD male rats, each weighing 220{tilde over ( )}250g, were acclimated for one week at 22-24° C. at a relative humidity of 60-80% with standard diet and water fed thereto. Thereafter, a solution of streptozotocin in saline was intravenously administered once at a dose of 50 mg/kg to the rats to induce type 1 diabetes. Four weeks after the induction of diabetes, rats were divided into groups according to blood sugar level. The extracts prepared in the above Examples were orally administered once a day for 8 weeks at predetermined doses to the rats, while a-lipoic acid and Gabapentin were used as positive controls at doses of 50 mg/kg and 100 mg/kg, respectively. Then, they were sacrificed to obtain the sciatic nerve and the salivary glands. The sciatic nerve and the salivary glands were processed in the same manner as in Test Example 1 and measured for NGF level by ELISA. The results are summarized in Table 7, below.

[0072] The ability of the extracts to alleviate the pain caused by diabetic neuropathy was examined. In this regard, rats with streptozotocin-induced diabetes were separated and orally administered with the extracts at a predetermined dose once a day for 14 weeks, with a-lipoic acid 50 mg/kg and Gabapentin 100 mg/kg serving as positive controls. A Randall-Sellito test was performed to measure a threshold response to pressure on a paw. The latent time taken to respond to a thermal pain was measured using a tail-flick test. The results are summarized in Table 8. In order to evaluate the neurodegeneration which had progressed with the onset of diabetic neuropathy, the sciatic nerve taken from the rats of each group was fixed with formalin and observed under an electron microscope to measure the size and thickness of the myelin sheath, and the results are summarized in Table 9.

TABLE-US-00007 TABLE 7 NGF Level in Sciatic Nerve of Type 2 Diabetes Rat Model (SD Male Rat) NGF Level Weight ratio of Sciatic Salivary Amount of material(kg) D. Rhizoma:D. Dose Nerve Gland Group D. Rhizoma D. nipponica nipponica (mg/kg) (pg/mg) (ng/mg) Normal 25.5 4.1 control 11.9 2.1 Ex. 1 1.55 0.45 3.5:1.sup.  100 35.4 3.8 C. Ex. 1 2 — — 100 13.1 2.0 C. Ex. 2 — 2 — 100 12.7 1.7 C. Ex. 3 1 1 1:1 100 19.2 1.9 C. Ex. 4 1.33 0.67 2:1 100 27.9 2.4 C. Ex. 5 1.67 0.33 5:1 100 25.1 2.9 C. Ex. 6 1.82 0.18 10:1  100 24.6 2.8 C. Ex. 7 0.67 1.33 1:2 100 19.1 3.3 C. Ex. 8 0.45 1.55 .sup. 1:3.5 100 19.8 2.9 C. Ex. 9 0.33 1.67 1:5 100 20.9 2.9 C. Ex. 10 0.18 1.82  1:10 100 16.4 2.5 α-Lipoic acid 50 13.8 2.8 Gabapentin 100 14.9 2.4

[0073] When administered at the same dosage, as can be seen in Table 7, the extract of Example 1 significantly increased the level of NGF in both the sciatic nerve and the salivary gland, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10

[0074] Thus, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica was proven to induce a significant increase of NGF in the target tissues (sciatic nerve, salivary gland) even in animals with type 1 diabetes.

TABLE-US-00008 TABLE 8 Assay for Pain Response in Type 1 Diabetes Rat Model (SD Male Rat) Weight ratio of Pressure Pain Latent Time to Amount of material(kg) D. Rhizoma:D. Dose Threshold Pain Reaction D. Rhizoma D. nipponica nipponica (mg/kg) (g) (sec) Normal 270.0 9.9 control 128.8 5.4 Ex. 1 1.55 0.45 3.5:1.sup.  100 191.7 11.4 C. Ex. 1 2 — — 100 130.1 6.3 C. Ex. 2 — 2 — 100 129.8 6.7 C. Ex. 3 1 1 1:1 100 169.0 8.0 C. Ex. 4 1.33 0.67 2:1 100 163.7 7.8 C. Ex. 5 1.67 0.33 5:1 100 138.0 7.4 C. Ex. 6 1.82 0.18 10:1  100 150.0 7.2 C. Ex. 7 0.67 1.33 1:2 100 153.5 7.9 C. Ex. 8 0.45 1.55 .sup. 1:3.5 100 157.0 7.6 C. Ex. 9 0.33 1.67 1:5 100 157.6 7.1 C. Ex. 10 0.18 1.82  1:10 100 129.2 7.5 α-Lipoic acid 50 150.0 7.4 Gabapentin 100 137.7 7.7

[0075] As can be seen in Table 8, when administered at the same dose, the extract of Example 1 significantly increased both the pressure pain threshold and the latent time to pressure response, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10.

[0076] An increase in threshold to mechanical stimuli delineates an increase in threshold to pain. In the tail flick test, an increase in latent time to pain response corresponds to an increase in threshold to thermal stimuli, indicating the alleviation of pain.

[0077] Hence, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica in accordance with the present invention exhibited a significant alleviative effect on pain, a symptom of diabetic peripheral neuropathy.

TABLE-US-00009 TABLE 9 Assay for Neurodegeneration in Type 1 Diabetes Rat Model (SD Male Rat) Neurodegeneration Dose Size of Myelin Thickness of Myelin (mg/kg) sheath (μm) sheath (μm) Normal 15.3 3.2 control 9.3 2.5 Ex. 1 30 13.6 3.2 100 13.8 2.9 α-Lipoic acid 50 11.2 2.6 Gabapentin 100 10.4 2.2

[0078] As is apparent from the data of Table 9, both size and thickness of the myelin sheath of the control became smaller, indicating the damage or degeneration of the sciatic nerve due to diabetic neuropathy. When administered with the extract of Example 1, both size and thickness of the myelin sheath of the rats were observed to become bigger.

[0079] Therefore, the extract of the present invention can ameliorate neurodegeneration, giving rise to nerve regeneration in target organs even in a histological aspect.

[0080] Consequently, the herb extract in accordance with the present invention can be applied to a pharmaceutical composition which can bring about a significant synergistic effect on the prevention and treatment of diabetic peripheral neuropathy in subjects with type 1 diabetes.

TEST EXAMPLE 3

[0081] Test for Effects on Pain, NGF Secretion, and Neurodegeneration in ICR Male Mice with Streptozotocin-Induced Type 1 Diabetes

[0082] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica, prepared in Example 1, was evaluated for effects on pain, in vivo NGF level and neurodegeneration in ICR male mice with type 1 diabetes.

[0083] ICR male mice, each weighing 25{tilde over ( )}30 g, were acclimated for one week at 22-24° C. at a relative humidity of 60-80%, with a standard diet and water fed thereto. Thereafter, a solution of streptozotocin in saline was intravenously administered once at a dose of 200 mg/kg to the mice to induce type 1 diabetes. Four weeks after the induction of diabetes, rats were divided into groups according to blood sugar level. The extracts prepared in the above Examples were orally administered once a day for 14 weeks at predetermined doses to the mice, while a-lipoic acid and Gabapentin were used as positive controls at a dose of 50 mg/kg and 100 mg/kg, respectively. Mice in each group were placed on a 58° C. hot plate, and examined for hyperalgesia to heat stimulus by measuring the latent time it took for the mice to feel a heat pain on their foot soles and jump. The results are summarized in Table 10.

[0084] Then, the mice were sacrificed to obtain the sciatic nerve and the salivary glands therefrom. The sciatic nerve and the salivary glands were processed in the same manner as in Test Example 1 and measured for NGF level by ELISA. The results are summarized in Table 11, below.

[0085] In order to evaluate the neurodegeneration which had progressed with the onset of diabetic neuropathy, the sciatic nerve taken from the mice of each group was fixed with formalin and observed under an electron microscope to measure the size and thickness of the myelin sheath, and the results are summarized in Table 12.

TABLE-US-00010 TABLE 10 Assay for Pain Response in Type 1 Diabetes Mouse Model (ICR Male Mouse) Dose Latent Time to Pain Response Group (mg/kg) on 58° C. Hot Plate (sec) Normal 55.3 control 25.2 Ex. 1 100 45.9 C. Ex. 1 100 26.6 C. Ex. 2 100 25.7 C. Ex. 3 100 35.6 C. Ex. 4 100 25.3 C. Ex. 5 100 26.0 C. Ex. 6 100 35.0 C. Ex. 7 100 32.2 C. Ex. 8 100 33.0 C. Ex. 9 100 26.8 C. Ex. 10 100 36.1 α-Lipoic acid 50 31.1 Gabapentin 100 33.0

[0086] An increase in latent time to pain response on the hot plate reflects an increase in pain threshold to thermal stimuli, indicating a reduction in pain. As can be seen in Table 10, the extract of Example 1 significantly increased the latent time to pain response on a hot plate, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of

[0087] Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10, demonstrating that the herb composition of the present invention has a significant alleviative effect on pain, a symptom of diabetic peripheral neuropathy.

TABLE-US-00011 TABLE 11 NGF Levels in Sciatic Nerve and Salivary Gland in Type 1 Diabetes Mouse Model (ICR Male Mouse) NGF Level Dose Sciatic Nerve Salivary Gland Group (mg/kg) (pg/mg) (ng/mg) Normal 9.7 226.1 control 3.9 101.8 Ex. 1 100 9.2 169.6 C. Ex. 1 100 4.1 107.2 C. Ex. 2 100 3.9 104.3 C. Ex. 3 100 6.2 118.7 C. Ex. 4 100 4.8 115.8 C. Ex. 5 100 4.0 121.0 C. Ex. 6 100 3.6 114.3 C. Ex. 7 100 6.2 122.7 C. Ex. 8 100 4.8 104.7 C. Ex. 9 100 4.1 104.7 C. Ex. 10 100 3.5 120.4 α-Lipoic acid 50 5.7 118.7 Gabapentin 100 5.5 117.1

[0088] As can be seen in Table 11, the extract of Example 1 significantly increased the level of NGF in both the sciatic nerve and the salivary gland, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10.

[0089] Thus, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica is proven to induce a significant increase of NGF in the target organs (sciatic nerve, salivary gland) in ICR male mice with type 1 diabetes as well as SD male rats with type 1 diabetes.

TABLE-US-00012 TABLE 12 Assay for Neurodegeneration in Type 1 Diabetes Mouse Model (ICR Male Mouse) Neurodegeneration Dose Size of Myelin Thickness of Myelin Group (mg/kg) sheath (μm) sheath (μm) Normal 14.3 3.4 control 9.1 2.0 Ex. 1 30 13.7 2.9 100 12.9 2.6

[0090] As is apparent from the data of Table 12, the myelin sheath of the control was decreased in both size and thickness, indicating the damage or degeneration of the sciatic nerve due to diabetic neuropathy. When administered with the extract of Example 1, mice were observed to have a myelin sheath which was increased in both size and thickness.

[0091] Therefore, the extract of the present invention can ameliorate neurodegeneration in ICR mice with type 1 diabetes as well as SD rats with type 1 diabetes, giving rise to nerve regeneration in target organs even in a histological aspect.

[0092] Consequently, the herb extract in accordance with the present invention can be applied to a pharmaceutical composition which can bring about a significant synergistic effect on the prevention and treatment of diabetic peripheral neuropathy in subjects with type 1 diabetes.

TEST EXAMPLE 4

[0093] Test for Effects on Pain and NGF Secretion in Male Mice with Type 2 Diabetes Induced Genetically (db/db Male Mice)

[0094] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica, prepared in Example 1, was evaluated for effects on pain and in vivo NGF level in db/db male mice with type 2 diabetes.

[0095] db/db Male mice, each weighing 40{tilde over ( )}45g, were acclimated for one week at 22-24° C. at a relative humidity of 60-80%, with a standard diet and water fed thereto. On the day of the 9th week after birth, the mice in which diabetes had been induced were selected. Four weeks later, mice suffering from type 2 diabetes were separated according to blood sugar level. The extracts prepared in the above Examples were orally administered once a day for 12 weeks at predetermined doses to the mice, while a-lipoic acid and Gabapentin were used as positive controls at a dose of 50 mg/kg and 100 mg/kg, respectively. Mice in each group were placed on a 58° C. hot plate, and examined for hyperalgesia to heat stimulus by measuring the latent time it took for the mice to feel a heat pain on their foot soles and jump. The results are summarized in Table 13.

[0096] Then, the mice were sacrificed to obtain the sciatic nerve therefrom. The sciatic nerve was processed in the same manner as in Test Example 1 and its NGF level was measured by ELISA. The results are summarized in Table 14, below.

TABLE-US-00013 TABLE 13 Assay for Pain Response in Type 2 Diabetes Mouse Model (db/db Male Mouse) Dose Latent Time to Pain Response Group (mg/kg) on 58° C. Hot Plate (sec) control 12.7 Ex. 1 100 28.0 C. Ex. 1 100 15.1 C. Ex. 2 100 14.9 C. Ex. 3 100 21.3 C. Ex. 4 100 21.0 C. Ex. 5 100 20.5 C. Ex. 6 100 19.3 C. Ex. 7 100 22.3 C. Ex. 8 100 17.6 C. Ex. 9 100 17.6 C. Ex. 10 100 16.8 α-Lipoic acid 50 15.4 Gabapentin 100 16.1

[0097] As can be seen in Table 13, the extract of Example 1 significantly increased the latent time to pain response on a hot plate, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10, demonstrating that the herb composition of the present invention has a significant alleviative effect on pain, a symptom of diabetic peripheral neuropathy.

TABLE-US-00014 TABLE 14 NGF Level in Sciatic Nerve of Type 2 Diabetes Mouse Model (db/db Male Mouse) Dose NGF Level in Sciatic Group (mg/kg) Nerve (pg/mg) control 6.2 Ex. 1 100 11.6 C. Ex. 1 100 6.5 C. Ex. 2 100 6.3 C. Ex. 3 100 8.4 C. Ex. 4 100 8.3 C. Ex. 5 100 7.8 C. Ex. 6 100 6.9 C. Ex. 7 100 8.1 C. Ex. 8 100 7.0 C. Ex. 9 100 7.1 C. Ex. 10 100 7.3 α-Lipoic acid 50 7.6 Gabapentin 100 7.2

[0098] As can be seen in Table 14, the extract of Example 1 significantly increased the level of NGF in the sciatic nerve, compared to the Dioscorea Rhizoma extract of Comparative Example 1, the Dioscorea nipponica extract of Comparative Example 2, or the extracts from mixtures of Dioscorea Rhizoma and Dioscorea nipponica of Comparative Examples 3 to 10.

[0099] Thus, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica is proven to induce a significant increase of NGF in the target organ even under the condition of type 2 diabetes.

[0100] Having the ability to significantly increase in vivo NGF levels, the herb extract in accordance with the present invention can be applied to a pharmaceutical composition which can bring about a significant synergistic effect on the prevention and treatment of diabetic peripheral neuropathy in subjects with type 1 or 2 diabetes.

TEST EXAMPLE 5

[0101] Test for Activity of Inducing Synthesis of NGF and Growth of Nerve Cells Using C6 Rat Glioma Cells and PC12 Nerve Cells

[0102] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica (w/w), prepared in Example 1, was examined for its ability to induce the synthesis of NGF in C6 rat glioma cells and the growth of PC12 rat nerve cells.

[0103] In order to evaluate the ability of the extract to induce the synthesis of NGF, C6 glioma cells were seeded at the same density per well into plates and incubated for 24 hours. The cells were washed with PBS, and cultured for 48 hours in fresh culture media supplemented with 50 μg/mL or 250 μg/mL herb extracts of Examples 1 or Comparative Examples 1 to 10. Then, the culture media were harvested and their NGF levels were quantitatively analyzed using ELISA. The results are summarized in Table 15.

[0104] For use in a test for ability to induce nerve cell growth, PC12 nerve cells were seeded at the same density per well into plates and incubated for 24 hours. The cells were washed with PBS and cultured for 48 hours in culture media supplemented with the herb extracts of Example 1 or Comparative Examples 1 to 10 or NGF. The growth of the cells was measured using MTS assay, and the results are summarized in Table 16.

TABLE-US-00015 TABLE 15 Promotive Effect on Synthesis of NGF Dose NGF Level Group (μg/ml) (% of control) control 100 Ex. 1 50 113 250 149 C. Ex. 1 50 — 250 107 C. Ex. 2 50 — 250 103 C. Ex. 3 50 108 250 110 C. Ex. 4 50 102 250 105 C. Ex. 5 50 101 250 110 C. Ex. 6 50 105 250 111 C. Ex. 7 50 102 250 122 C. Ex. 8 50 107 250 128 C. Ex. 9 50 104 250 119 C. Ex. 10 50 101 250 123

[0105] As can be seen in Table 15, the extract of Example 1 significantly increased the level of NGF in the C6 cells, compared to the extract of Comparative Example 1 (from Dioscorea Rhizoma alone), the extract of Comparative Example 2 (from Dioscorea nipponica alone) or the extracts of Comparative Examples 3 to 10 (from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios).

[0106] Thus, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica is proven to maximally induce the synthesis of NGF.

TABLE-US-00016 TABLE 16 Proliferative Effect on Nerve Cells Dose Proliferative Effect on Group (μg/ml) Nerve Cell (% of control) control 100 NGF  2 (ng/ml) 112 50 (ng/ml) 118 Ex. 1 1 111 30 117 C. Ex. 1 1 — 30 101 C. Ex. 2 1 — 30 100 C. Ex. 3 1 101 30 105 C. Ex. 4 1 98 30 103 C. Ex. 5 1 100 30 101 C. Ex. 6 1 99 30 102 C. Ex. 7 50 104 250 106 C. Ex. 8 50 100 250 103 C. Ex. 9 50 99 250 101 C. Ex. 10 50 104 250 102

[0107] As can be seen in Table 16, the extract of Example 1 significantly promoted the growth of PC12 nerve cells, compared to the extract of Comparative Example 1 (from Dioscorea Rhizoma alone), the extract of Comparative Example 2 (from Dioscorea nipponica alone) or the extracts of Comparative Examples 3 to 10 (from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios).

[0108] Thus, the herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica in accordance with the present invention is proven to have a significant proliferative effect on nerve cells.

TEST EXAMPLE 6

[0109] Test for Phosphorylation of NGF Receptor in PC12 Nerve Cells

[0110] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica, prepared in Example 1, was evaluated for its ability to promote the phosphorylation of NGF receptors in PC12 rat nerve cells.

[0111] For use in this test, PC12 nerve cells were seeded at the same density per well into plates and incubated for 24 hours. The cells were washed with PBS and cultured for 48 hours in culture media supplemented with the herb extracts of Example 1 or Comparative Examples 1 to 10 or NGF. Thereafter, phosphorylated TrkA (NGF receptor) were detected using Western blotting, and quantitatively analyzed on the basis of the thickness of the detected bands. The results are summarized in Table 17.

[0112] NGF signaling starts with the binding of NGF to its receptor (Trk-A), which results in the phosphorylation of the receptor. Thus, an increased level of phosphorylated NGF receptors means the amplification of the NGF signaling pathway.

[0113] As is apparent from data of Table 17, below, the extract of Example 1 induced a significant increase in the phosphorylation of NGF receptors, compared to the extract of Comparative Example 1 (from Dioscorea Rhizoma alone), the extract of Comparative Example 2 (from Dioscorea nipponica alone) or the extracts of Comparative Examples 3 to 10 (from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios).

TABLE-US-00017 TABLE 17 Promotive Effect on Phosphorylation of NGF Receptor Dose Relative Amount of NGF Combined Extract Phosphorylated NGF Group (ng/ml) (μg/ml) (% of control) Control 100 NGF 2 — 241 50 — 337 Ex. 1 + NGF 2 200 309 500 389 C. Ex. 1 + NGF 2 200 — 500 232 C. Ex. 2 + NGF 2 200 — 500 229 C. Ex. 3 + NGF 2 200 268 500 299 C. Ex. 4 + NGF 2 200 255 500 287 C. Ex. 5 + NGF 2 200 254 500 274 C. Ex. 6 + NGF 2 200 248 500 277 C. Ex. 7 + NGF 2 200 251 500 268 C. Ex. 8 + NGF 2 200 239 500 287 C. Ex. 9 + NGF 2 200 249 500 296 C. Ex. 10 + NGF 2 200 244 500 280

TEST EXAMPLE 7

[0114] Test for Activity to Induce the Growth of Neurites in Rat-Derived DRG Nerve Cells

[0115] The herb extract of a mixture of 3.5:1 Dioscorea Rhizoma:Dioscorea nipponica, prepared in Example 1, was evaluated for activity to promote the growth of neurites in DRG (Dorsal root ganglion) nerve cells obtained from rat embryos.

[0116] For use in this test, DRG nerve cells were seeded at the same density into plates and incubated for 24 hours. Thereafter, the cells were washed with. PBS and cultured for 48 hours in a culture medium in the absence or presence of a combination of the extract of Example 1 or Comparative Examples 1 to 10, and NGF. The nerve cells in each group were observed under a microscope to measure the area of the neurites grown therein. Average values of the measurements are given in Table 18.

[0117] An increase in neurite area indicates that the nerve cells are activated to grow neurites. As can be seen in Table 18, below, the extract of Example 1 significantly increased a neurite area, compared to the extract of Comparative Example 1 (from Dioscorea Rhizoma alone), the extract of Comparative Example 2 (from Dioscorea nipponica alone) or the extracts of Comparative Examples 3 to 10 (from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios).

TABLE-US-00018 TABLE 18 Promotive Effect on Formation of Neurites from DRG Nerve Cell Dose NGF Combined Extract Neurite Area of DRG Group (ng/ml) (μg/ml) Nerve Cell (μm.sup.2) Control 149 NGF 2 — 235 50 — 461 Ex. 1 + NGF 2 30 291 100 502 C. Ex. 1 + NGF 2 30 — 100 234 C. Ex. 2 + NGF 2 30 — 100 236 C. Ex. 3 + NGF 2 30 264 100 288 C. Ex. 4 + NGF 2 30 259 100 271 C. Ex. 5 + NGF 2 30 241 100 294 C. Ex. 6 + NGF 2 30 232 100 301 C. Ex. 7 + NGF 2 30 269 100 288 C. Ex. 8 + NGF 2 30 257 100 279 C. Ex. 9 + NGF 2 30 241 100 266 C. Ex. 10 + NGF 2 30 239 100 248

[0118] Taken together, the data obtained from Test Examples 1 to 7 demonstrate that the extract of Example 1 can significantly induce the elevation of in vivo NGF levels, the growth of nerve cells and neurites, the alleviation of pain, a symptom of diabetic neuropathy, and the prevention of histological neurodegeneration (the promotion of nerve regeneration), compared to the extract of Comparative Example 1 (from Dioscorea Rhizoma alone), the extract of Comparative Example 2 (from Dioscorea nipponica alone) or the extracts of Comparative Examples 3 to 10 (from mixtures of Dioscorea Rhizoma and Dioscorea nipponica at other weight ratios) and thus can be applied to pharmaceutical compositions and health aid foods for the prevention and treatment of diabetic peripheral neuropathy.

[0119] Below, a description will be given of illustrative, non-limiting examples of formulations containing the herb extract from a mixture of Dioscorea Rhizoma and Dioscorea nipponica.

FORMULATION EXAMPLE 1

Preparation of Injection

[0120] Extract of Example 1 . . . 100 mg [0121] Sodium metabisulfite . . . 3.0 mg [0122] Methylparaben . . . 0.8 mg [0123] Propylparaben 0.1 mg [0124] Sterile water for injection . . . q.s.

[0125] To a mixture of the ingredients was added sterile water to form a total volume of 2 mL, and the solution was loaded to a 2 mL ampule and sterilized to give an injection.

FORMULATION EXAMPLE 2

Preparation of Tablet

[0126] Extract of Example 1 . . . 200 mg [0127] Lactose . . . 100 mg [0128] Starch . . . 100 mg [0129] Mg stearate . . . q.s.

[0130] The ingredients were mixed and compressed into a tablet using a tableting method.

FORMULATION EXAMPLE 3

Preparation of Capsule

[0131] Extract of Example 1 . . . 100 mg [0132] Lactose . . . 50 mg [0133] Starch . . . 50 mg [0134] Talc . . . 2 mg [0135] Mg Stearate . . . q. s.

[0136] The ingredients were mixed and loaded to a gelatin capsule according to a typical method to afford a capsule.

FORMULATION EXAMPLE 4

Preparation of Liquid

[0137] Extract of Example 1 . . . 1000 mg [0138] Sugar . . . 20 g [0139] Isomerase . . . 20 g [0140] Lemon Flavor . . . q. s. [0141] Purified water added to form a total volume of 100 mL.

[0142] The above ingredients were mixed, loaded into a 100 mL brown vial and sterilized to afford a liquid formulation.

[0143] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.