Indian Patents. 270519:A POLY HERBAL COMPOSITION FOR WOUND HEALING AND A PROCESS FOR PRODUCING THE SAME

Title of Invention	A POLY HERBAL COMPOSITION FOR WOUND HEALING AND A PROCESS FOR PRODUCING THE SAME
Abstract	A polyherbal composition for wound healing comprising: 2.5- 7.5% of Alternanthera brasiliana and 2.5 - 7.5% of Achyranthes aspera.

Full Text	-1- FIELD OF INVENTION: This invention relates to a polyherbal composition for wound healing and a process for producing the same. BACKGROUND OF THE INVENTION: In general, we try to treat wounds with the help of home remedies and in most of the cases they get success. In case of complications the rural natives consult the traditional healers. The natives living in urban areas initially try antibiotics but when problem persists, they consult the healers or use traditional medicine. The traditional healers informed that the cases having this problem are increasing from past two or three decades. The old wounds indicate the poor resistance of human body. If problem occurs at regular interval then there must be some basic health problem needing enough attention. In general, the healers suggest the natives to give emphasis on blood purification. At the start of rainy season, they suggest the natives to prepare globules (size of Ber fruits) from Neem leaves and take one globule in a day up to one week. This simple use make the body resistant round the year. They warn that seven days means seven days, it should not be taken more than a week. They also suggest to take bath by using herbal decoction and juice. Kadu Pani (please read previous articles) is one of these herbal baths. The healers blame -2- the modern life for the cause of this problem. Now it is a fashion here, that seeing any old wound, the registered practitioners recommend pathological test for diabetes. In reference literatures on weed science, Alternanthera is mentioned as common weed. In India, our researchers invest thousands of Rupees, in search of Agrochemicals to manage this weed. Every year the farmers are dumping these Agrochemicals to manage the weed but the war between them are still in good progress. In Chhattisgarh, Alternanthera grows as waste plant in crop fields and wastelands but the natives and traditional healers use it as medicinal weed. It is surprising to know that this herb is in trade from many parts of Chhattisgarh as medicinal herb. The recommendation of experts are with the farmers but they are not ready to use weedicides. Through the ethnobotanical surveys conducted in different parts of Chhattisgarh. According to the reference literatures, in many states of India, this herb is under cultivation as vegetable crop. This is really depressing that one place this is used as medicine, in other place the natives are using it as vegetable and at the same time our researchers are investing time, money and effort to destroy it. A comparative study of the traditional herbal knowledge of the islanders of the Indian Ocean and the folk of India shows that both use about seventy species of plants in Indian medicine. Whereas some uses are common among the folk of -3- two regions, many traditional uses among the islanders are unique. Comparative data on such species is presented. Details of uses, dosages mixture plants or other materials are given for some unique uses/species. The advantage of substituting underground parts of plants with aerial parts use discussed. Comparative study of traditional medicinal uses of plants between the folk of different regions, countries or ethnic groups bring out valuable information on credibility, similarities, uniqueness and interesting aspects.. This communication deals with such study on the islanders of the Indian Ocean and the folk in India. A large part of the ocean lying between equator and tropics of Capricorn, and Africa and Australia is called the Indian Ocean. The landmass of Indian region lies just North of this ocean, and thus the floras of India and islands of Indian Ocean have hundreds of common species of plants. Major islands of the Indian Ocean such as Madagascar, Mauritius, Comoros, Mascarenes have received considerable attention of botanists and the flora of the region is reasonably well documented. The indigenous people who have their own traditional pharmacopoeia inhabit the islands predominantly. A genus of herbs or small shrubs, distributed throughout the tropical and sub-tropical regions. About nine species occur in India. -4-OBJECTS OF THE INVENTION: An object of this invention is to propose a polyherbal composition for wound healing. Another object of this invention is to propose a process for producing a polyherbal composition. Further, object of this invention is to propose a polyherbal composition which exhibits synergism and results in the wound healing activity. Still further object of this invention is to propose a polyherbal composition which is obtained from a perennial plant which is available throughout the year. DETAILED DESCRIPTION OF THE INVENTION: Wound healing activity of Alternanthera brasiliana and Achyranthes aspera (5%each): The epithelialization of the wounds in case of the rats treated with methanolic fraction of Alternanthera brasiliana and Achyranthes aspera and were found to be quite earlier followed by standard drug Himax and the control group. In case of Alternanthera brasiliana and Achyranthes aspera scar formation is observed on 21st day after complete healing, whereas in case of standard group as well as the control group it was absent till 28th day post wounding. -5- Table 1. Wound healing activity of Alternanthera brasiliana and Achyranthes aspera in rats (5%). Post wounding day Wound area (mm^), Mean ± SE Gr.l Gr 11 Grill Gr.IV Oday 32.3± 0.00 32.3±0.00 32.3 ± 0.00 32.3±0.00 7 day 28.58±1.53 23.63 ±6.63 16.45 ±1.53 27.62±7.35 14 day 13.32±0.55 3.77± 0.52 4.2±0.16 9.88±2.75 21 day 6.13±0.50 - - 2.68±0.21 28 day 2.48± 0.26 - - 0.32±0.10 = Achyranthes N= 6. Gr. I = control. Gr II = Alternanthera brasiliana ,Gr. aspera, Gr. IV = Standard (Himax). ANOVA TABLE Source d.f. MSS F Group 3 1.0506 22.209 Days 4 26.744 394.396 Interaction groups x Days 12 7.304 107.712** Error 100 0.678 Total 119 (P Group, days and interaction effect are highly significant. CD. test indicates among the all 4 groups, Group-ll (Achyranthes) is most effective one followed by Group -I (Alternanthera) Value for CD=0.131. -6- Table 2. Percentage wound contraction in rats treated with Alternanthera brasiliana (5%) and Achyranthes aspera (5%). Post wounding day Wound contraction in terms of percentage. Gr1 Gr.ll Grill Gr.IV Oday 0.00 0.00 0.00 0.00 7 day 11.52 26.84 49.07 14.49 14 day 58.76 88.33 86.99 69.41 21day 81.02 100.00 100.00 91.70 28 day 92.32 100.00 100.00 99.01 N= 6. Gr. I = control. Gr II = Alternanthera brasiliana, Gr. aspera, Gr. IV = Standard (Himax). = Achyranthes Table 3. Wound healing activity of Alternanthera brasiliana(2.5%) + Achyranthes aspera (2.5%) and Alternathera brasiliana(5%) and Achyranthes aspera(5%). Table 3. Wound healing activity of . Alternanthera brasiliana + Achyranthes aspera (2.5%) and Alternathera (5%) and Achyranthes (5%). Post Wound area in cm * wounding days Gr.-I Gr.-I I Gr.-ll Gr.-IV Gr.V f 0 3 23±0.00 3. 23±0. 00 3 .23±0. 00 3 .23±0 00 3 .23±0 00 7 1 007±0.06 2. 36±0. 16 1 ,65±0. 08 2 .77±0 33 2 .86±0 08 14 ro 25+0.55 0. 38+0. 04 0 .42±0. 02 0 .99±0 10 1 .33±0 05 21 - - - 0 .27±0 03 0 .63±0 04 28 - - - 0 .03±0 02 0 .25±0 03 Gr. -I Alternanthera 2.5%+ Achyranthes. 2.5% Gr. -IV Standard (Himax) Gr.- II Alternanthera 5% Gr. -VControl Gr. -Ill Achyranthes 5% -7- ANOVA TABLE Source d.f. SS MSS Variance ratio (F) Group 4 9.3070 2.3268 17.17 Days 4 223.3862 55.8466 412.15 Interaction groups x Days 16 9.9619 0.6226 4.60 Error 125 16.9361 0.1355 Total 149 259.5912 (P Treatment Gr. - I a Gr. Illa b Gr. IIb Gr. IV c Gr. Vc Group means 0.89 1.059 1.194 1.459 1.564 1. All the effects due to different groups, days and their interaction come out as highly significant 2. CD. test indicates Treatment Gr.-I, II,Ml are significantly different from Gr.IV (standard) and gr. V (control) i.e. they are more effective as compared to Gr.IV and V s performances 3. CD test also indicate that I. Gr. t and Gr. Ill can be treated as alike II. Grill and Gr. II can be treated as alike But, if we have to make a selection between Gr. I and Gr.ll , then we should select Gr. I treatment because performance of the Gr. I is significantly than that of Gr. II treatment. -8- Table 4. Wound healing activity of Alternanthera brasiliana + Achyranthes aspera+ Azadirachta indica and Ocimum sanctum (1.25%each) and Alternanthera brasiliana 1.25% +Achyranthes aspera 1.25% + Azadirachta indica 2.5% in rats. Wound healing activity of Alternanthera brasiliana + Achyranthes aspera+ Azadirachta indica and Ocimum sanctum (1.25%each) and Alternanthera brasiliana 1.25% +Achyranthes aspera 1.25% + Azadirachta indica 2.5% in rats. Post Wound area in cm 2 wounding days Gr.-I Gr.-ll Gr.-lll Gr.-IV 0 16.1±0.00 16.1±0.00 16.1 ±0.00 16.1 ±0.00 7 8.4±0.04 8.2±0.05 11.3±0.04 12.3±0.05 14 1.05±0.02 1.9±0.03 7.2±0.01 8.4±0.04 21 - _ 4.0±0.05 5.1±0.05 28 - - 0.5±0.01 1.3±0.03 Gr. -I Alternanthera 1.25%+Achyranthes 1.25% + Azadirachta 1.25% + Ocimum 1.25% Gr.- II Alternanthera 1.25% + Achyranthes . 1.25% + Azadirachta 2.5% Gr. -Ill Standard (Himax) Gr. -IV Control. -9- ANOVA TABLE Source d.f. MSS Variance ratio (F) Group 3 96.4532 150.47 Days 4 975.3527 1521.61 Interaction groups x Days 12 13.8960 21.68 Error 100 0.6410 Total 119 - (P CD. Value 0.405 Results: Treatment groups are come out as significant 1. CD. test shows- a. Group I , II and III are significantly different from the control group. They shows better performance than the control group b. Again, Group I and II are equally effective. Both are significantly different from the standard group (Group-Ill). Both of them can be considered as the most effective treatment group among the four. B. Incision wound model: In this model, tensile strength of the breaking tissue after treatment in healing wound was measured in tensiometer. (Instron ) by the method of Lee, 1968. Tensile strength of Alternathera brasiliana (5%), Achyranthes aspera (5%) Alternathera brasiliana, +Achyranthes aspera (2.5%) in rats. -10- Table 5: Tensile strength of Alternathera brasiliana (5%), Achyranthes aspera (5%), Alternathera brasiliana, +Achyranthes aspera (2.5%) in rats. Treatment group Mean ± SE Grl Achyranthes 5% 4.063 a ±0 339 Gr.ll Alternathera 5% 4.861 a ±0.664 Grill. Achyranthes2.5%+ Alternathera 2.5% 6.772 ±0.372 Gr.IV Standard (Himax) 3.778 a ±1.092 Gr.V Control 4.267 a ±0.639 Treatment bearing same superscript do not differ significantly (P For determination of wound healing activity following extract/fractions were used as they were found to be the most active fractions in the anti microbial study. 1. Methanolic fractions of Achyranthes aspera (5 and 7.5%) 2. Methanolic fraction of Alternanthera brasiliana (5 and 7.5%) 3. Methanolic fractions of Achyranthes aspera (2.5%) and Methanolic fraction of Alternanthera brasiliana (2.5%) EXAMPLE: Animal : Sprague Dawley rats (150-200g) of either sex were used from the departmental animal house. Animals were housed individually under standard environmental condition (25± 3° C and light dark cycles). Food and water were given ad lib. Additionally, we can also claim the method for preparing the formulation : Answer: -11- Drug formulation : i. Five gram each of the fractions were mixed with Vaseline to make 100 g of ointment for 5% ointment w/w. ii. For 7.5% ointment, 7.5 g of the extract was mixed with Vaseline to make 100 g of the ointment, iii. Again 2.5 g each of the extracts were mixed in Vaseline base to get 100 g of 2.5% ointment. Q : In this connection we would then require the method in details alongwith all process step and parameters. Answer: Evaluation of wound healing activity of Alternanthera brasiliana and Achyranthes aspera: For evaluation wound healing activity excision wound model is used. 1. Excision wound model: The animals were randomly assigned to four groups of six animals each. They were depilated at the desired site before wounding. The animals were starved for 12 hours prior to wounding. The animals were anaesthetized as described by Morton and Malone, (1972) under light ether anaesthesia. The skin of the impressed area was excised to full thickness to obtain a wound area of about 2.5 cm. The first group served as control and topically applied vehicle only , second and third group received methanol extract of Alternanthera brasiliana (5% or 7.5 % or 2.5%), Achyrantehs aspera (5% or 7.5% or 2.5% combination) respectively and -12- fourth group received the standard drug Himax, by topical application once daily from the day of creation of wound i.e. 0 day till complete epithelialization. The parameters studied were: i. Wound closure (wound closure was measured at regular intervals of time to see the percentage of wound closure) and ii. Epithelialization time (the epithelialization time indicates the formation of new epithelial tissue to cover the wound). iii. The percentage of wound closure was recorded on 7,14,21 and 28 post wounding till complete re-epithelialization. Refer to Table 1,2, 3 and 4 with statistical analysis. 2. Incision wound model: Animal: Sprague Dawley rats of either sex weighing between 100-150 g were maintained and bred in the departmental animal house and used for the experiment. The animals were fasted overnight before the start of the experiment. In this model, 6 cm long paravertebral incisions were made through the full thickness of the skin on either side of the vertebral column of the rats as described by Ehrlich and Hunt et al., The wounds were closed with interrupted sutures of 1 cm apart. The animals were divided into different groups of 6 animals each. The animals of group l were left untreated and considered as the Control, the Group II and Group III received 50 mg of ointment prepared from -13- methanol leaf extract of Alternathera brasiliana and Achyranthes aspera. Group IV served as reference standard and treated with Himax (50 mg).The ointment was topically applied once a day. The sutures were removed on the 8th post wound day. Parameter measured : The skin breaking strength of the wound was measured on the 10th day day as described by the method of Lee, 1968. The result is shown in Table 5. -14-WE CLAIM: 1. A polyherbal composition for wound healing comprising: 2.5- 7.5% of Alternanthera brasiliana and 2.5 - 7.5% of Achyranthes aspera. 2. The polyherbal composition as claimed in claim 1, wherein best results are obtained when each of Alternanthera brasiliana and Achyranthes aspera are present in 2.5% each. 3. A method for preparing a polyherbal composition comprising: mixing the alcoholic extract of Alternanthera brasiliana with alcoholic extract of Achyranthes aspera in a ratio of 1:1. 4. The method as claimed in claim 3 wherein the alcohol used is methanol. A polyherbal composition for wound healing comprising: 2.5- 7.5% of Alternanthera brasiliana and 2.5 - 7.5% of Achyranthes aspera.

Full Text

-1-
FIELD OF INVENTION:
This invention relates to a polyherbal composition for wound healing and a process for producing the same.
BACKGROUND OF THE INVENTION:
In general, we try to treat wounds with the help of home remedies and in most of the cases they get success. In case of complications the rural natives consult the traditional healers. The natives living in urban areas initially try antibiotics but when problem persists, they consult the healers or use traditional medicine. The traditional healers informed that the cases having this problem are increasing from past two or three decades. The old wounds indicate the poor resistance of human body. If problem occurs at regular interval then there must be some basic health problem needing enough attention.
In general, the healers suggest the natives to give emphasis on blood purification. At the start of rainy season, they suggest the natives to prepare globules (size of Ber fruits) from Neem leaves and take one globule in a day up to one week. This simple use make the body resistant round the year. They warn that seven days means seven days, it should not be taken more than a week. They also suggest to take bath by using herbal decoction and juice. Kadu Pani (please read previous articles) is one of these herbal baths. The healers blame
-2-
the modern life for the cause of this problem. Now it is a fashion here, that seeing any old wound, the registered practitioners recommend pathological test for diabetes.
In reference literatures on weed science, Alternanthera is mentioned as common weed. In India, our researchers invest thousands of Rupees, in search of Agrochemicals to manage this weed. Every year the farmers are dumping these Agrochemicals to manage the weed but the war between them are still in good progress. In Chhattisgarh, Alternanthera grows as waste plant in crop fields and wastelands but the natives and traditional healers use it as medicinal weed. It is surprising to know that this herb is in trade from many parts of Chhattisgarh as medicinal herb. The recommendation of experts are with the farmers but they are not ready to use weedicides. Through the ethnobotanical surveys conducted in different parts of Chhattisgarh. According to the reference literatures, in many states of India, this herb is under cultivation as vegetable crop. This is really depressing that one place this is used as medicine, in other place the natives are using it as vegetable and at the same time our researchers are investing time, money and effort to destroy it.
A comparative study of the traditional herbal knowledge of the islanders of the Indian Ocean and the folk of India shows that both use about seventy species of plants in Indian medicine. Whereas some uses are common among the folk of
-3-
two regions, many traditional uses among the islanders are unique. Comparative data on such species is presented. Details of uses, dosages mixture plants or other materials are given for some unique uses/species. The advantage of substituting underground parts of plants with aerial parts use discussed.
Comparative study of traditional medicinal uses of plants between the folk of different regions, countries or ethnic groups bring out valuable information on credibility, similarities, uniqueness and interesting aspects.. This communication deals with such study on the islanders of the Indian Ocean and the folk in India.
A large part of the ocean lying between equator and tropics of Capricorn, and Africa and Australia is called the Indian Ocean. The landmass of Indian region lies just North of this ocean, and thus the floras of India and islands of Indian Ocean have hundreds of common species of plants. Major islands of the Indian Ocean such as Madagascar, Mauritius, Comoros, Mascarenes have received considerable attention of botanists and the flora of the region is reasonably well documented. The indigenous people who have their own traditional pharmacopoeia inhabit the islands predominantly.
A genus of herbs or small shrubs, distributed throughout the tropical and sub-tropical regions. About nine species occur in India.
-4-OBJECTS OF THE INVENTION:
An object of this invention is to propose a polyherbal composition for wound healing.
Another object of this invention is to propose a process for producing a polyherbal composition.
Further, object of this invention is to propose a polyherbal composition which exhibits synergism and results in the wound healing activity.
Still further object of this invention is to propose a polyherbal composition which is obtained from a perennial plant which is available throughout the year.
DETAILED DESCRIPTION OF THE INVENTION:
Wound healing activity of Alternanthera brasiliana and Achyranthes aspera (5%each):
The epithelialization of the wounds in case of the rats treated with methanolic fraction of Alternanthera brasiliana and Achyranthes aspera and were found to be quite earlier followed by standard drug Himax and the control group. In case of Alternanthera brasiliana and Achyranthes aspera scar formation is observed on 21st day after complete healing, whereas in case of standard group as well as the control group it was absent till 28th day post wounding.
-5-
Table 1. Wound healing activity of Alternanthera brasiliana and Achyranthes aspera in rats (5%).

Post wounding day Wound area (mm^), Mean ± SE

Gr.l Gr 11 Grill Gr.IV
Oday 32.3± 0.00 32.3±0.00 32.3 ± 0.00 32.3±0.00
7 day 28.58±1.53 23.63 ±6.63 16.45 ±1.53 27.62±7.35
14 day 13.32±0.55 3.77± 0.52 4.2±0.16 9.88±2.75
21 day 6.13±0.50 - - 2.68±0.21
28 day 2.48± 0.26 - - 0.32±0.10
= Achyranthes
N= 6. Gr. I = control. Gr II = Alternanthera brasiliana ,Gr. aspera, Gr. IV = Standard (Himax).
ANOVA TABLE

Source d.f. MSS F
Group 3 1.0506 22.209**
Days 4 26.744 394.396**
Interaction groups x Days 12 7.304 107.712**
Error 100 0.678
Total 119
(P Group, days and interaction effect are highly significant.
CD. test indicates among the all 4 groups, Group-ll (Achyranthes) is most
effective one followed by Group -I (Alternanthera) Value for CD=0.131.
-6-
Table 2. Percentage wound contraction in rats treated with Alternanthera brasiliana (5%) and Achyranthes aspera (5%).
Post wounding day Wound contraction in terms of percentage.

Gr1 Gr.ll Grill Gr.IV
Oday 0.00 0.00 0.00 0.00
7 day 11.52 26.84 49.07 14.49
14 day 58.76 88.33 86.99 69.41
21day 81.02 100.00 100.00 91.70
28 day 92.32 100.00 100.00 99.01
N= 6. Gr. I = control. Gr II = Alternanthera brasiliana, Gr. aspera, Gr. IV = Standard (Himax).
= Achyranthes
Table 3. Wound healing activity of Alternanthera brasiliana(2.5%) + Achyranthes aspera (2.5%) and Alternathera brasiliana(5%) and Achyranthes aspera(5%).
Table 3. Wound healing activity of . Alternanthera brasiliana + Achyranthes aspera (2.5%) and Alternathera (5%) and Achyranthes (5%).

Post Wound area in cm *
wounding days Gr.-I Gr.-I I Gr.-ll Gr.-IV Gr.V f
0 3 23±0.00 3. 23±0. 00 3 .23±0. 00 3 .23±0 00 3 .23±0 00
7 1 007±0.06 2. 36±0. 16 1 ,65±0. 08 2 .77±0 33 2 .86±0 08
14 ro 25+0.55 0. 38+0. 04 0 .42±0. 02 0 .99±0 10 1 .33±0 05
21 - - - 0 .27±0 03 0 .63±0 04
28 - - - 0 .03±0 02 0 .25±0 03
Gr. -I Alternanthera 2.5%+ Achyranthes. 2.5% Gr. -IV Standard (Himax) Gr.- II Alternanthera 5% Gr. -VControl Gr. -Ill Achyranthes 5%
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ANOVA TABLE
Source d.f. SS MSS Variance ratio (F)
Group 4 9.3070 2.3268 17.17**
Days 4 223.3862 55.8466 412.15**
Interaction groups x Days 16 9.9619 0.6226 4.60**
Error 125 16.9361 0.1355
Total 149 259.5912
(P Treatment Gr. - I a Gr. Illa b Gr. IIb Gr. IV c Gr. Vc
Group means 0.89 1.059 1.194 1.459 1.564
1. All the effects due to different groups, days and their interaction come out
as highly significant
2. CD. test indicates
Treatment Gr.-I, II,Ml are significantly different from Gr.IV (standard) and gr. V (control) i.e. they are more effective as compared to Gr.IV and V s performances 3. CD test also indicate that
I. Gr. t and Gr. Ill can be treated as alike
II. Grill and Gr. II can be treated as alike
But, if we have to make a selection between Gr. I and Gr.ll , then we should select Gr. I treatment because performance of the Gr. I is significantly than that of Gr. II treatment.
-8-
Table 4. Wound healing activity of Alternanthera brasiliana + Achyranthes aspera+ Azadirachta indica and Ocimum sanctum (1.25%each) and Alternanthera brasiliana 1.25% +Achyranthes aspera 1.25% + Azadirachta indica 2.5% in rats.
Wound healing activity of Alternanthera brasiliana + Achyranthes aspera+ Azadirachta indica and Ocimum sanctum (1.25%each) and Alternanthera brasiliana 1.25% +Achyranthes aspera 1.25% + Azadirachta indica 2.5% in rats.

Post Wound area in cm 2
wounding days Gr.-I Gr.-ll Gr.-lll Gr.-IV
0 16.1±0.00 16.1±0.00 16.1 ±0.00 16.1 ±0.00
7 8.4±0.04 8.2±0.05 11.3±0.04 12.3±0.05
14 1.05±0.02 1.9±0.03 7.2±0.01 8.4±0.04
21 - _ 4.0±0.05 5.1±0.05
28 - - 0.5±0.01 1.3±0.03
Gr. -I Alternanthera 1.25%+Achyranthes 1.25% + Azadirachta 1.25% + Ocimum
1.25%
Gr.- II Alternanthera 1.25% + Achyranthes . 1.25% + Azadirachta 2.5% Gr. -Ill Standard (Himax) Gr. -IV Control.
-9-
ANOVA TABLE

Source d.f. MSS Variance ratio
(F)
Group 3 96.4532 150.47**
Days 4 975.3527 1521.61**
Interaction groups x Days 12 13.8960 21.68**
Error 100 0.6410
Total 119 -
(P CD. Value 0.405
Results: Treatment groups are come out as significant
1. CD. test shows-
a. Group I , II and III are significantly different from the control group. They
shows better performance than the control group
b. Again, Group I and II are equally effective. Both are significantly different
from the standard group (Group-Ill). Both of them can be considered as
the most effective treatment group among the four.
B. Incision wound model:
In this model, tensile strength of the breaking tissue after treatment in healing wound was measured in tensiometer. (Instron ) by the method of Lee, 1968. Tensile strength of Alternathera brasiliana (5%), Achyranthes aspera (5%) Alternathera brasiliana, +Achyranthes aspera (2.5%) in rats.
-10-
Table 5: Tensile strength of Alternathera brasiliana (5%), Achyranthes aspera (5%), Alternathera brasiliana, +Achyranthes aspera (2.5%) in rats.

Treatment group Mean ± SE
Grl Achyranthes 5% 4.063 a ±0 339
Gr.ll Alternathera 5% 4.861 a ±0.664
Grill. Achyranthes2.5%+ Alternathera 2.5% 6.772 ±0.372
Gr.IV Standard (Himax) 3.778 a ±1.092
Gr.V Control 4.267 a ±0.639
Treatment bearing same superscript do not differ significantly (P For determination of wound healing activity following extract/fractions were used as they were found to be the most active fractions in the anti microbial study.
1. Methanolic fractions of Achyranthes aspera (5 and 7.5%)
2. Methanolic fraction of Alternanthera brasiliana (5 and 7.5%)
3. Methanolic fractions of Achyranthes aspera (2.5%) and Methanolic
fraction of Alternanthera brasiliana (2.5%)
EXAMPLE:
Animal : Sprague Dawley rats (150-200g) of either sex were used from the departmental animal house. Animals were housed individually under standard environmental condition (25± 3° C and light dark cycles). Food and water were
given ad lib.
Additionally, we can also claim the method for preparing the formulation :
Answer:
-11-
Drug formulation :
i. Five gram each of the fractions were mixed with Vaseline to make 100
g of ointment for 5% ointment w/w. ii. For 7.5% ointment, 7.5 g of the extract was mixed with Vaseline to
make 100 g of the ointment, iii. Again 2.5 g each of the extracts were mixed in Vaseline base to get
100 g of 2.5% ointment.
Q : In this connection we would then require the method in details
alongwith all process step and parameters.
Answer: Evaluation of wound healing activity of Alternanthera brasiliana and Achyranthes aspera: For evaluation wound healing activity excision wound model is used.
1. Excision wound model:
The animals were randomly assigned to four groups of six animals each. They
were depilated at the desired site before wounding. The animals were starved for
12 hours prior to wounding. The animals were anaesthetized as described by
Morton and Malone, (1972) under light ether anaesthesia. The skin of the
impressed area was excised to full thickness to obtain a wound area of about 2.5
cm.
The first group served as control and topically applied vehicle only , second and
third group received methanol extract of Alternanthera brasiliana (5% or 7.5 % or
2.5%), Achyrantehs aspera (5% or 7.5% or 2.5% combination) respectively and
-12-
fourth group received the standard drug Himax, by topical application once daily from the day of creation of wound i.e. 0 day till complete epithelialization. The parameters studied were:
i. Wound closure (wound closure was measured at regular intervals of
time to see the percentage of wound closure) and ii. Epithelialization time (the epithelialization time indicates the formation
of new epithelial tissue to cover the wound).
iii. The percentage of wound closure was recorded on 7,14,21 and 28 post wounding till complete re-epithelialization.
Refer to Table 1,2, 3 and 4 with statistical analysis.
2. Incision wound model:
Animal: Sprague Dawley rats of either sex weighing between 100-150 g were
maintained and bred in the departmental animal house and used for the
experiment.
The animals were fasted overnight before the start of the experiment.
In this model, 6 cm long paravertebral incisions were made through the full
thickness of the skin on either side of the vertebral column of the rats as
described by Ehrlich and Hunt et al., The wounds were closed with interrupted
sutures of 1 cm apart. The animals were divided into different groups of 6
animals each. The animals of group l were left untreated and considered as the
Control, the Group II and Group III received 50 mg of ointment prepared from
-13-
methanol leaf extract of Alternathera brasiliana and Achyranthes aspera. Group IV served as reference standard and treated with Himax (50 mg).The ointment was topically applied once a day. The sutures were removed on the 8th post wound day.
Parameter measured : The skin breaking strength of the wound was measured on the 10th day day as described by the method of Lee, 1968. The result is shown in Table 5.
-14-WE CLAIM:
1. A polyherbal composition for wound healing comprising:
2.5- 7.5% of Alternanthera brasiliana and 2.5 - 7.5% of Achyranthes aspera.
2. The polyherbal composition as claimed in claim 1, wherein best results are
obtained when each of Alternanthera brasiliana and Achyranthes aspera
are present in 2.5% each.
3. A method for preparing a polyherbal composition comprising:
mixing the alcoholic extract of Alternanthera brasiliana with alcoholic extract of Achyranthes aspera in a ratio of 1:1.
4. The method as claimed in claim 3 wherein the alcohol used is methanol.

A polyherbal composition for wound healing comprising:
2.5- 7.5% of Alternanthera brasiliana and 2.5 - 7.5% of Achyranthes aspera.

Documents:

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Patent Number

270519

Indian Patent Application Number

426/KOL/2007

PG Journal Number

01/2016

Publication Date

01-Jan-2016

Grant Date

29-Dec-2015

Date of Filing

20-Mar-2007

Name of Patentee

ASSAM AGRICULTURAL UNIVERSITY

Applicant Address

KHANAPARA, GUWAHATI-781022,

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. CHANDANA CHOUDHURY BARUA	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA
2	RAJEEV KUMAR SHARMA	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA
3	ISWAR CHANDRA BARUA	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA
4	APURBA CHAKRABORTY	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA
5	SEEMA PEGU	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA
6	ANIMESH DEKA	DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, COLLEGE OF VETERINARY SCIENCE, ASSAM AGRICULTURAL UNIVERSITY, KHANAPARA, GUWAHATI-781022, INDIA

PCT International Classification Number

C07D

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA