The present invention relates to development of analgesic, anti-inflammatory and an anti-arthritic herbal composition and preparation method thereof.
Background of the Invention
References which are cited in the present disclosure are not necessarily prior art and therefore their citation does not constitute an admission that such references are prior art in any jurisdiction. All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual or patent application was specifically and individually indicated to be incorporated by reference.
Several patents have been issued for topical medicaments to relieve the pain and inflammation caused by arthritis and other similar diseases. For example, CN101282737A teaches a herbal pharmaceutical formulation comprising by the Korea's Chinese angelica root extract that uses ethanol, distilled water or the preparation of its mixture extraction, herb aArtemisiae Scopariae extract for regenerating cartilaginous tissue and treating osteoarthritis.
Another patent, KR20110035100A teaches a formulation for the treatment of osteoarthritis, comprising a herbal drug as a main component, thaw skin extract bioconverted by Sacchromyces cerevisae BH02 or Sacchromyces exiguus BH12.
Other inventions for similar application use many herbal ingredients or plants. Patent WO2003059370A1 uses a pharmaceutical composition essentially comprising a complex herbal extract of Chaenomelis Fructus, Achyranthis Radix, Acanthopanax, Phlomidis Radix, Gentianae Radix, Clematidis Radix and a pharmaceutical^ acceptable carrier thereof as an active ingredient for treating and preventing arthritic diseases.

Another invention, WO2006061675A1 is a synergistic herbal composition useful for the treatment of arthritis, said composition comprising pharmaceutically effective amount of extracts obtained from the plants Terminalia chebula, Pluchea lanceolata, Desmodium gangeticum, Vitex negundo and Zingiber officinale.
While such externally-applied medicaments may have some effects on the treatment of arthritis, the effects are only seen insofar as the ability of the medicament to penetrate the superficial tissue into the muscles and joints when applied externally. It is therefore an object of the present invention to further improve an herbal medicament for arthritis and anti-inflammatory and analgesic activities. There is a revival of interest with herbal-based medicine due to the increasing realization of the health hazards associated with the indiscriminate use of modern medicine. The demand for plant-based medicines, health products, pharmaceuticals, food supplement, cosmetics etc are increasing in both developing and developed countries, due to the growing recognition that the natural products are non-toxic, have less side effects and easily available at affordable prices.
The primary object of the present invention is to provide a formulation and composition for herbal extract medication.
Another object of the present invention is to provide herbal drug of Asparagus officinalis for the analgesic, anti-inflammatory and anti-arthritic activity.
Another object of the present invention is to provide herbal extracts of Asparagus officinalis as the analgesic, anti-inflammatory and anti-arthritic disease.
Another object of the present invention is to provide herbal extracts of the arial part of Asparagus officinalis.
Another object of the present invention is to provide herbal extracts of Asparagus officinalis

and Asparagus racemosus, Asparagus sprengeri, and Asparagus acutifolius all members of the same genus to evaluate the analgesic, anti-inflammatory and anti-arthritic activity.
Another object of the present invention is to provide herbal extracts of arial part ofAsparagus officinalis and petroleum ether, or ethanol, or water to evaluate the analgesic, anti-inflammatory and anti-arthritic activity.
These and other objects and advantages of the present invention will become readily apparent from the following detailed description.
Summary of Invention
This summary is not a comprehensive overview of the disclosure and does not reflect the main/essential features of the establishment or specify the scope of the establishment. Its sole purpose is to present some of the concepts presented here in a simpler way as a precursor to more detailed explanations presented later.
The present invention relates to be used for the herbal composition of analgesic, anti-Inflammatory and an anti-arthritic. More specifically, said composition has anti-inflammatory activity, anti-arthritic and analgesic activities, and therefore can be used as the medicine of treatment arthritis.
In some embodiments of the present invention, the formulation comprises extracts of Asparagus officinalis and Asparagus racemosus, Asparagus sprengeri, and Asparagus acutifolius all members of the same genus. Specifically, the formulation comprises extracts of Asparagus officinalis.
In a first preferred embodiment of this aspect of the present invention, the very first step is collecting and washing the arial part of Asparagus officinalis with water and air drying and

making the power of this. The powdered drug was defatted with petroleum ether, ethanol and then extracted with water in Soxhlet apparatus.
The extraction step comprises refluxing arial part of the Asparagus officinalis with petroleum ether to produce an Extract 1; with ethanol to produce an Extract 2; with water to form an Extract 3 in Soxhlet apparatus. These extracts can then be used individually or in combination. In a preferred embodiment, a combined extract is formed by individually extracts.
In some embodiments of the present invention, the formulation dried under vacuum to result into solid form.
In the teachings of the present invention, any of these extraction methods can be followed by a purification process, preferably by passage through a thin layer chromatography for separation of compounds.
The present invention also provides, in another aspect, a method of treating arthritis inflammatory and analgesic activities in a mammal comprising orally administering a therapeutically effective amount of the composition given above or obtained by the method taught above.
The present invention also teaches a composition described herein for use in a medicament for the treatment of arthritis, inflammatory and analgesic in a mammal. Thus, the present invention also relates in a further aspect to a medicament or health supplement that can be applied to the body to exert its effects in deeper tissues in the treatment of arthritis and inflammatory conditions.
These and other aspects of the embodiments herein will be better appreciated and understood when considered in concurrence with the following explanation and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating

preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
Detailed Description of the invention
These embodiments are described in sufficient detail to enable those skilled in the art to practice
the embodiments and all technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which this invention belongs.
The various embodiments of the present invention provide the herbal composition of analgesic,
anti-Inflammatory and an anti-arthritic.
In some embodiments of the invention, a formulation comprising the one plant extract is
prepared.
Plant extract is obtained from the herb Asparagus officinalis and Asparagus racemosus,
Asparagus sprengeri, and Asparagus acutifolius all members of the same genus.
A formulation comprising the extracts of plant materials Asparagus officmalis, Asparagus
racemosus, Asparagus sprengeri, and Asparagus acutifolius thereof as an active ingredient
effective for analgesic, antiarthritis and antiinflammation.
The pharmaceutical composition further comprise the extract of herb selected from the group
consisting of Asparagus officinalis, Asparagus racemosus, Asparagus sprengeri, and
Asparagus acutifolius thereof as an active ingredient for preventing and treating arthritic
diseases.
The formulation wherein, Asparagus officinalis is selected from the group consisting of
Asparagus racemosus, Asparagus sprengeri, and Asparagus acutifolius.
The formulation is in a form suitable for oral administration.
The formulation is selected from the group consisting of powder, capsule, tablet, liquid and

caplet.
A formulation effective for analgesic, antiarthritis and antiinflammation comprising therapeutically effective amounts of extract of herb and petroleum ether or ethanolic or water in the following range by weight: Asparagus officinalis - petroleum ether, 75-300 mg/kg, Asparagus officinalis - ethanol, 75-300 mg/kg and Asparagus officinalis - water, 75-300 mg/kg. A method for preparing the formulation of claim 1, comprising:
a. collecting the arial part of Asparagus officinalis and washing with water;
b. drying arial part of plant materials obtained from step a and making powder;
c. defatting the powdered drug obtained from step b with more than once with suitable
liquids to obtain extracts in Soxhlet apparatus;
d. drying extract under vacuum to result into solid form;
e. subjecting the extract to thin layer chromatography for separation of compounds; and
f conducting the study to determine the effects of the extracts paw edema, WBC's level
in blood serum and histopathological changes paw joints.
The method, wherein the Asparagus officinalis extracting step further comprises extracting the
Asparagus officinalis residue with a petroleum ether to produce another extract.
The method, wherein the Asparagus officinalis extracting step further comprises extracting the
Asparagus officinalis residue with an alcohol to produce another extract.
The method, wherein the Asparagus officinalis extracting step further comprises extracting the
Asparagus officinalis residue with water to produce another extract.
The formulation, wherein the formulation is conducted to evaluate the analgesic activity and
significant effect found at 180 min with aqueous, alcoholic and petroleum ether extracts.
The formulation, wherein the formulation is conducted to evaluate the anti- inflammatory effect
2-6 hours with aqueous, alcoholic and petroleum ether extracts.
The formulation of claim 1, wherein the formulation is conducted to evaluate the anti- arthritic

effect within day 14 -21 with aqueous, alcoholic and petroleum ether extracts.
Examples 1
Extraction Methods
Generalized Extraction Method
The generalized extraction process comprises reducing the size of the herbal materials (for
example by pulverizing) followed by extraction by refluxing with a suitable solvent.
The extracts are then dried under vacuum. The components of the present invention were
subjected to various combinations of extraction methods by petroleum ether, ethanol and water
in order to determine the optimal process with the highest desired pharmacological effects.
At any point in the process, the intermediate products of any step may be subjected to
concentration, clarification or purification steps. Thereafter, some of the extracts were further
purified and tested for improved efficacy.
The Asparagus officinalis were collected, washed with water, air dried and then pulverized to
a coarse powder, further defatted with petroleum ether, ethanol and then extracted with water
in Soxhlet apparatus. The extract was dried under vacuum to result into solid form.
An extract of the formulation obtained from the process described above may be further
purified. One way of increasing the purity of the desired is to subject the extract to one or more
separation techniques in which the unwanted components are reduced, thus increasing the
relative abundance of the desired components in the extract.
A preferred separation technique is thin layer chromatography for separation of compounds
and to get the desired components but not the unwanted components under certain conditions.
Examples 2
A first implementation example of the invention to obtain the extract followed by an example
of a purification process to illustrate how the invention may be practiced.

The following procedure is for the preparation of an extract in the proportion of the formulation of the first implementation. The dried herb was separately pulverized into coarse powders.
The Asparagus officinalis was pulverized and defatted with petroleum ether. The petroleum ether extract was then filtered. The filtrate was then dried by vacuum to produce the Extract 1 in the range of 75 - 300 mg/kg (w/w).
The Asparagus officinalis was extracted and defatted with ethanol to produce Extract 2 in the range of 75 - 300 mg/kg (w/w). The filtrate was then dried by vacuum.
The Asparagus officinalis was defatted with water and then filtered. The filtrate was then dried by vacuum to produce the Extract 3 in the range of 75 - 300 mg/kg (w/w).
The analgesic effects of above extracts of the present invention were investigated in acute analgesic models induced by paw edema, WBC's level in blood serum and histopathological changes paw joints. The significant effect was observed at 180 min with aqueous, alcoholic and petroleum ether extracts. Table 1: Effect of Asparagus officinalis extracts in analgesic activity on rats

Groups (n = 6) 0 min (s) After 30 min (s) After 60 min (s) After 90 min (s) After 120 min (s) After 180 min (s)
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 1.84 ±0.35 1.76 ±0.42 1.87 ±0.38 1.85 ±0.38 1.93 ±0.24 1.62 ±0.42
Group 2 (Diclofenac, 10 mg/kg) 1.69 ±0.18 1.84±0.19 (4.74) 2.71 ±0.52 c (45.05) 4.54 ±0.37 ' (145.76) 5.84 ± 0.44" (203.29) 3.63 ± 0.42" (124.97)
Group 3 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 1.73 ±0.38 1.83 ±0.32 (3.79) 1.86 ±0.40 (-0.62) 2.09 ±0.24 (13.18) 2.21 ±0.41 (14.62) 1.91 ±0.18 (18.27)
Group 4 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 1.66 ±0.23 1.99±0.18 (13.08) 2.13 ±0.19 (14.18) 2.17 ±0.49 (17.33) 2.51 ±0.32 c (30.19) 2.60 ± 0.34' (60.68)
Group 5 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 1.73 ±0.25 1.73 ±0.22 (-1.71) 1.96 ±0.21 (4.91) 2.08 ±0.19 (12.55) 2.54 ±0.32 c (31.66) 2.96 ± 0.46' (83.49)
Group 6 (Asparagus officinalis, Ethanol, 75 mg/kg) 1.76 ±0.23 1.79±0.10 (1.80) 1.95 ±0.38 (4.19) 2.20 ±0.18 (19.13) 2.16 ±0.24 (12.11) 2.23 ± 0.35 c (37.87)
Group 7 (Asparagus officinalis, Ethanol, 150 mg/kg) 1.74 ±0.34 1.91 ±0.14 (8.34) 2.07 ±0.41 (10.97) 2.40 ± 0.26 c (29.78) 2.29 ±0.21 (18.60) 2.66 ± 0.37" (64.60)
Group 8 (Asparagus officinalis, Ethanol, 300 mg/kg) 1.77 ±0.34 2.02 ±0.16 (14.88) 2.19±0.19 (16.95) 2.19 ±0.25 (18.50) 2.35 ±0.28 (21.80) 2.88 ± 0.26" (78.22)
Group 9 (Asparagus officinalis, Water, 75 mg/kg) 1.70 ±0.22 1.94±0.19 (10.33) 1.97 ±0.50 (5.26) 2.04 ±0.27 (10.65) 2.17 ±0.39 (17.82) 2.36 ± 0.27 c (46.23)
Group 10 (Asparagus officinalis, Water, 150 mg/kg) 1.67 ±0.32 1.95 ±0.24 (11.00) 2.00 ±0.17 (6.87) 2.19± 0.18 (18.32) 2.27 ±0.20 (17.99) 2.60 ± 0.23 " (61.20)
Group 11 (Asparagus officinalis, Water, 300 mg/kg) 1.74 ±0.17 1.86 ±0.14 (5.97) 2.08 ±0.35 (11.42) 2.57 ±0.41 c (39.08) 2.43 ± 0.28 (25.87) 2.92 ± 0.19' (80.50)
F Value 0.1938 1.056 2.598 34.03 71.89 16.49
P Value 0.9960 0.4112 0.0117 O.0001 O.0001 O.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), aJP<0.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
The anti-inflammatory effects of above extracts of the present invention were investigated in acute inflammatory models induced by paw edema, WBC's level in blood serum and histopathological changes paw joints. The significant effect was observed anti- inflammatory effect 2 hour to 6 hours with aqueous, alcoholic and petroleum ether extracts.
Table 2: Effect of Asparagus officinalis extracts in Acute Inflammation (Using plethysmometer) activity on rats

(n=6) Pre- Induction hour 1st hour 2nd hour 3rd hour 4th hour 6th hour
(Normal Control, Tween 80 v 0.5%w/v CMC in water ) 0.690 ± 0.023 (-2.73) 0.695 ± 0.019(1.65) 0.672 ± 0.015 " (23.24) 0.667 ±0.022 (26.74)a 0.663 ± 0.023 (29.18)a 0.663 ± 0.019" (23.90)

Group 2 (Inflammation Control) 0.672 ±0.028 0.707 ± 0.022 0.875 ±0.019 0.910 ±0.011 0.937 ±0.022 0.872 ±0.028
Group 3 (Diclofenac, 10 mg/kg) 0.682 ±0.023 (-1.49) 0.705 ± 0.018 (0.24) 0.723 ± 0.016" (17.33) 0.752 ± 0.038" (17.40) 0.735 ± 0.019" (21.53) 0.702 ± 0.017" (19.50)
Group 4 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 0.678 ± 0.033 (-0.99) 0.707 ± 0.010 (0.00) 0.732 ± 0.026' (16.38) 0.768 ± 0.029' (15.57) 0.753 ± 0.024' (19.57) 0.737 ± 0.019' (15.49)
Group 5 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 0.682 ±0.029 (-1.49) 0.713 ± 0.020 (-0.94) 0.760 ± 0.011 ' (13.14) 0.785 ± 0.019' (13.74) 0.740 ± 0.023 ' (21.00) 0.717±0.016a (17.78)
Group 6 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 0.675 ± 0.027 (-0.50) 0.715 ± 0.005 (-1.18) 0.742 ± 0.015 ' (15.24) 0.798 ± 0.042' (12.27) 0.722 ± 0.029' (22.95) 0.733 ± 0.022' (15.87)
Group 7 (Asparagus officinalis, Ethanol, 75 mg/kg) 0.667 ±0.027 (0.74) 0.702 ± 0.015 (0.71) 0.743 ± 0.024" (15.05) 0.778 ± 0.033 " (14.47) 0.745 ± 0.018" (20.46) 0.713 ± 0.022" (18.16)
Group 8 (Asparagus officinalis, Ethanol, 150 mg/kg) 0.678 ±0.019 (-0.99) 0.715 ± 0.016 (-1.18) 0.740 ± 0.021 " (15.43) 0.788 ± 0.023 " (13.37) 0.748 ± 0.015 " (20.11) 0.725 ± 0.019" (16.83)
Group 9 (Asparagus officinalis, Ethanol, 300 mg/kg) 0.668 ± 0.029 (0.50) 0.713 ± 0.022 (-0.94) 0.757 ± 0.029" (13.52) 0.808 ± 0.012" (11.17) 0.752 ± 0.015 " (19.75) 0.732 ± 0.020" (16.06)
Group 10 (Asparagus officinalis, Water, 75 mg/kg) 0.668 ±0.025 (0.50) 0.713 ± 0.012 (-0.94) 0.755 ± 0.019" (13.71) 0.793 ± 0.016" (12.82) 0.773 ± 0.016" (17.44) 0.715 ± 0.019" (17.97)
Group 11 (Asparagus officinalis, Water, 150 mg/kg) 0.672 ±0.031 (0.50) 0.718 ± 0.020 (-1.65) 0.740 ± 0.014' (15.43) 0.753 ± 0.015 ' (17.22) 0.743 ± 0.012' (20.74) 0.702 ± 0.018' (19.50)
Group 12 (Asparagus officinalis, Water, 300 mg/kg) 0.675 ± 0.029 (-0.50) 0.723 ± 0.018 (-2.36) 0.788 ± 0.017' (9.90) 0.818 ± 0.017' (10.07) 0.782 ± 0.019' (16.55) 0.715 ± 0.015 ' (17.97)
F Value 0.3819 1.186 35.30 29.37 60.09 37.85
P Value 0.9582 0.3162 O.0001 O.0001 O.0001 O.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), a.P<0.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
The anti-arthritic activity of Asparagus officinalis evaluated and it was observed statistically
significant effect on body weight with aqueous and alcoholic extracts
Table 3: Effect of Asparagus officinalis extracts on body weights of Arthritic animals

Groups (n = 6) Dayl Day 7 Day IS Day 21
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 128.96±10. 72 (-0.03) 179.67±10. 99 (-5.46) 244.19±13. 66 (4.25) 299.30±12.00 c (9.74)
Group 2 (Arthritic Control) 128.99±10. 20 190.05±13. 41 234.24±13. 40 272.74±8.44
Group 3 (Methotrexate, 0.5 mg/kg) 129.10±9.7 5 (0.08) 181.75±12. 26 (-4.37) 236.55±15. 15 (0.98) 294.53±12.33 (7.99)
Group 4 (Prednisolone, 5 mg/kg) 129.43±9.0 6 (0.34) 182.93±9.6
7 (-3.75) 236.80±10. 34(1.09) 286.80±12.49 (5.16)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 129.70±6.9 7(0.55) 189.53±7.8 4 (-0.27) 242.50±10. 91 (3.53) 292.88±12.89 (7.39)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 129.71±7.0 0 (0.56) 188.57±8.5 7 (-0.77) 238.25±15. 54(1.71) 282.87±20.50 (3.71)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 129.68±6.9 6(0.53) 187.54±10. 12 (-1.32) 234.47±17. 94(0.10) 292.39±18.28 (7.21)

Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 129.75±6.8 3 (0.59) 189.76±11. 09 (-0.15) 240.12±20. 13(2.51) 289.28±20.00 (6.07)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 129.77±6.8 3 (0.60) 192.10±5.4 6(1.08) 243.53±6.3 9(3.97) 299.04±9.20 c (9.65)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 129.73±6.8 9(0.57) 189.80±8.5 0(-0.13) 242.64±11. 59(3.59) 289.56±14.32 (6.17)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 129.76±6.8 4(0.59) 177.73±11. 82 (-6.48) 230.76±15. 92(1.48) 272.39±22.40 (0.13)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 129.84±6.9 5 (0.66) 199.14±6.0 5 (4.79) 254.41±10. 06 c (8.61) 294.82±18.86 (8.10)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 129.90±6.9 7(0.71) 190.37±8.1 0(0.17) 240.08±13. 13 (2.49) 312.99±11.16b (14.76)
F Value 0.01032 2.070 1.109 2.957
P Value >0.9999 0.0315 0.3687 0.0025
It was observed that water, alcoholic and petroleum ether extract has significant anti- arthritic effect. Effect starts with day 14 and final observation was taken on day 21. Anti- arthritic activity was evaluated using water displacement method with plethysmometer at different days.
Table 4: Effect of Asparagus officinalis extracts in Arthritis (Using plethysmometer) activity on rats

Groups (n = 6) 1st Day 7hDay 14th Day 21s'Day
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 0.74 ± 0.02 0.94 ± 0.02' (49.09) 1.26 ± 0.06' (48.11) 1.50 ± 0.09' (40.41)
Group 2 (Arthritic Control) 0.75 ± 0.03 1.84 ±0.03 2.42 ±0.33 2.51 ±0.26
Group 3 (Methotrexate, 0.5 mg/kg) 0.74 ± 0.05 1.84 ±0.05 (0.09) 1.92±0.17a (20.65) 1.80 ± 0.16" (28.33)
Group 4 (Prednisolone, 5 mg/kg) 0.73 ± 0.01 1.82±0.10 (0.73) 1.77±0.12a (26.77) 1.70 ± 0.15 " (32.18)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 0.73 ± 0.02 1.82 ±0.04 (1.13) 2.20 ± 0.07 (9.36) 2.23 ± 0.03 c (11.41)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 0.73 ± 0.02 1.84 ±0.03 (0.00) 2.02 ± 0.08' (16.66) 1.91 ± 0.08' (23.95)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 0.74 ± 0.02 1.83 ±0.02 (0.54) 1.98 ± 0.09' (18.44) 1.92 ± 0.12' (23.56)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 0.74 ± 0.02 1.82 ±0.03 (1.18) 2.22 ±0.16 (8.40) 2.08 ± 0.11 ' (17.05)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 0.75 ± 0.02 1.84 ±0.02 (0.09) 2.10± 0.11b (13.42) 2.12±0.19a (16.19)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 0.74 ± 0.03 1.80 ±0.03 (1.91) 1.87 ± 0.06" (22.64) 1.85 ± 0.08" (26.34)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 0.74 ± 0.02 1.83 ±0.04 (0.36) 2.17 ± 0.07 c (10.25) 2.09 ± 0.10" (16.72)

Group 12 (Asparagus officinalis, Water, 150 mg/kg) 0.73 ± 0.03 1.84 ±0.04 (0.00) 2.03 ± 0.15 " (16.10) 1.94 ± 0.17" (22.89)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 0.73 ± 0.02 1.83 ±0.04 (0.27) 1.86 ± 0.09" (23.33) 1.77 ± 0.09" (29.66)
F Value 0.4824 204.9 24.41 20.68
P Value 0.9180 O.0001 O.0001 O.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), TO.0001, hP<0.00\,c P<0.05
compared with negative control group, ns: non-significant.
It was observed that water, alcoholic and petroleum ether extract has significant anti- arthritic effect. Effect starts with day 10 and final observation was taken on day 21. Anti- arthritic activity was evaluated by taking paw diameter with Vernier caliper.
Table 5: Effect of Asparagus officinalis extracts in Arthritis (Using Vernier calliper) activity on rats

Groups (n = 6) Is'Day (mm) 7th Day (mm) Iff" Day (mm) 15th Day (mm) 20th Day (mm)
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 4.33 ± 0.23 4.45 ± 0.26' (48.32) 5.15 ±0.40 a (48.27) 5.37 ±0.21 a(46.14) 5.54±0.
13 ' (43.82)
Group 2 (Arthritic Control) 4.29 ±0.12 7.84 ± 0.87 9.95 ± 0.59 9.98 ± 0.27 9.86 ± 0.40
Group 3 (Methotrexate, 0.5 mg/kg) 4.31 ±0.28 7.48 ±
0.44
(4.65) 8.14±0.82 a (18.14) 7.22 ± 0.450' (27.68) 6.83 ± 0.21' (30.78)
Group 4 (Prednisolone, 5 mg/kg) 4.40 ± 0.26 7.55 ±
0.40
(3.76) 7.90 ± 0.24 ' (20.58) 6.84 ±0.41 ' (31.44) 6.61 ± 0.50 a (32.97)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 4.17 ±0.26 7.43 ± 0.33 (5.21) 8.75 ±0.71 c (12.05) 8.24 ±0.58 a (17.46) 8.02 ± 0.44' (18.69)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 4.21 ± 0.07 7.41 ± 0.16 (5.46) 8.65 ±0.27 c (13.06) 7.65 ±0.58 ' (23.35) 7.27 ± 0.27a (26.25)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 4.27 ±0.27 7.48 ±
0.16
(4.59) 8.80 ±0.78 c (11.51) 7.52±0.51 a (24.61) 7.21 ± 0.52' (26.87)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 4.22 ±0.09 7.51 ±
0.49
(4.23) 8.45 ±0.90 b (15.02) 7.52 ±0.64 ' (24.66) 7.67 ± 0.42a (22.19)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 4.21 ±0.10 7.34 ±
0.37
(6.44) 8.86 ±0.74 c (10.93) 7.35 ±0.42 a (26.34) 7.41 ± 0.48' (24.84)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 4.22 ±0.12 7.37 ± 0.32 (5.99) 8.05 ±0.59 ' (19.02) 7.11 ±0.40 ' (28.75) 6.91 ± 0.34a (29.91)

Group 11 (Asparagus officinalis, Water, 75 mg/kg) 4.21 ±0.15 7.55 ± 0.29 (3.72) 8.26 ± 0.40 b (16.98) 7.50 ±0.39 ' (24.79) 7.45 ± 0.34a (24.45)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 4.19±0.17 7.68 ± 0.52 (2.13) 8.17±0.50 a (17.86) 7.55 ±0.42 a (24.29) 7.44 ± 0.59' (24.52)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 4.21 ±0.14 7.43 ± 0.40 (5.27) 8.31 ±0.63 b (16.44) 7.44 ±0.71 ' (25.41) 6.49 ± 0.33 a (34.21)
F Value 0.7459 24.77 18.18 26.36 36.83
P Value 0.7018 O.0001 0.0001 O.0001 O.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), a.P<0.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
It was observed that water, alcoholic and petroleum ether extract has significant anti- arthritic activity. Visual scorings were given based on walking, inflammation, joint deformities and pain assessment using Grimace scale (0= Normal to 5= Severe).
Table 6: Effect of Asparagus officinalis extracts in Arthritis (Using Arthritis Visual Score) activity on rats

Groups (n = 6) 1st Day $hDay WDay If Day 2ffhDay
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 0.0000 0.00 ± 0.00' (100) 0.00 ± 0.00' (100) 0.00 ± 0.00' (100) 0.00 ± 0.00' (100)
Group 2 (Arthritic Control) 0.0000 2.67 ± 0.52 4.50 ± 0.55 4.50 ± 0.55 4.50 ± 0.55
Group 3 (Methotrexate, 0.5 mg/kg) 0.0000 2.83 ± 0.41 (-6.25) 4.33 ± 0.52 (3.70) 2.67 ± 0.52a (40.74) 2.17± 0.75 a (51.85)
Group 4 (Prednisolone, 5 mg/kg) 0.0000 2.67 ± 0.52 (0.00) 3.50 ± 0.55 c (22.22) 2.17± 0.41' (51.85) 0.83 ± 0.75 ' (81.48)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 0.0000 2.67 ± 0.52 (0.00) 4.67 ± 0.52 (-3.70) 4.17± 0.75 (7.41) 3.50 ±
0.55
(22.22)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 0.0000 2.83 ± 0.41 (-6.25) 4.67 ± 0.52 (-3.70) 3.33 ± 0.52c (25.93) 2.67 ± 0.52' (40.74)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 0.0000 3.00 ± 0.00 (-12.50) 4.67 ± 0.52 (-3.70) 2.83 ± 0.41a (37.04) 2.33 ± 0.82 a (48.15)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 0.0000 2.67 ± 0.52 (0.00) 4.83 ± 0.41 (-7.40) 3.67 ±
0.82
(18.52) 3.83 ±
0.75
(14.81)

Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 0.0000 3.00 ± 0.00 (-12.50) 4.83 ± 0.41 (-7.40) 3.17± 0.41b (29.63) 3.50 ±
0.55
(22.22)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 0.0000 3.00 ± 0.00 (-12.50) 4.67 ± 0.52 (-3.70) 2.33 ± 0.52' (48.15) 2.00 ± 0.63 ' (55.56)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 0.0000 2.83 ± 0.41 (-6.25) 4.83 ± 0.41 (-7.40) 3.17± 0.41b (29.63) 3.67 ±
0.52
(18.52)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 0.0000 2.83 ± 0.41 (-6.25) 4.50 ± 0.55 (0.00) 3.33 ± 0.52c (25.93) 2.83 ± 0.75 b (37.04)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 0.0000 3.00 ± 0.00 (-12.50) 4.67 ± 0.52 (-3.70) 2.50 ± 0.55 a (44.44) 1.50 ± 0.55 a (66.67)
F Value 28.27 44.18 26.71 25.19
P Value 0.0001 0.0001 0.0001 0.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), aJP<0.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
On last day animals were sacrificed and Spleen was collected and spleen weight was recorded we observed ethanolic extract is significantly able to retain the spleen weight in normal range whereas some activity was also noticed with petroleum ether and alcoholic extracts also. Table 7: Effect of Asparagus officinalis extracts in Arthritis Animal Spleen Weight

Groups (n = 6) Spleen Weight (g)
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 0.73 ± 0.08 c (19.67)
Group 2 (Arthritic Control) 0.91 ±0.08
Group 3 (Methotrexate, 0.5 mg/kg) 0.75 ± 0.09 c (18.28)
Group 4 (Prednisolone, 5 mg/kg) 0.52 ± 0.08 " (43.38)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 0.82 ±0.09 (10.30)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 0.70 ± 0.14 c (23.59)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 0.72 ± 0.06 c (20.80)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 0.69 ± 0.11 c (25.08)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 0.71 ± 0.09 c (22.09)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 0.62 ± 0.07 ' (32.03)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 0.76 ±0.13 (17.37)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 0.73 ± 0.10 c (20.20)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 0.67 ± 0.08 b (26.94)

F Value 5.702
P Value <0.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), a.P<0.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
Rat serum was analyzed for RA Factor aqueous, ethanol and petroleum ether extracts were
observed with statistically significant anti- arthritic activity.
Table 8: Effect of Asparagus officinalis extracts on RA Factor in Arthritis.

Groups (n = 6) IU/mL
Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 7.60 ± 1.15 ' (65.51)
Group 2 (Arthritic Control) 22.03 ±0.59
Group 3 (Methotrexate, 0.5 mg/kg) 13.57 ± 4.19b (38.43)
Group 4 (Prednisolone, 5 mg/kg) 11.17±5.35a(49.32)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 14.03 ± 2.01c (36.31)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 9.70 ± 3.05 ' (55.98)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 13.63 ± 4.34b (38.12)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 11.23 ± 2.06 ' (49.02)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 15.40 ± 3.65 c (30.11)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 7.93 ± 2.67 " (63.99)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 16.83 ±0.06 (23.60)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 12.93 ± 7.01b (41.30)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 9.40 ± 1.81 " (57.34)
F Value 7.776
P Value <0.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), TO.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
During arthritis it is noticed that WBC's increase. All the extracts were found very effective and statistically significant and helped in keeping the WBC's in range with normal group. Table 9: Effect of Asparagus officinalis extracts on WBC's in Arthritis.

Groups (n = 6)

Cells/cumm

Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v)) 6066.67 ± 1795.36 s (58.06)
Group 2 (Arthritic Control) 14466.67 ±750.56
■ ■'■■ .-; .• (■.'-. .'■'.- -.'■-. •-.'.'-.. " .■■ ■■■..• ' .•' 8200.00 ± 2271.56 s (43.32)
Group 4 (Prednisolone, 5 mg/kg) 5033.33 ± 702.38 s (65.21)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg) 8400.00 ± 3508.56 s (41.94)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg) 4566.67 ± 1553.49 s (68.43)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg) 5333.33 ± 1123.98 s (63.13)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg) 6933.33 ± 2064.78 s (52.07)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg) 6133.33 ± 680.69 s (57.60)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg) 3300.00 ± 173.21s (77.19)
Group 11 (Asparagus officinalis, Water, 75 mg/kg) 8233.33 ± 862.17 s (43.09)
Group 12 (Asparagus officinalis, Water, 150 mg/kg) 5600.00 ± 1135.78 s (61.29)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 5066.67 ± 2369.25 s (64.98)
F Value 16.02
P Value <0.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple ra
test. The values are expressed as mean ± SD for each group (n=6), TO.0001, hP<0.00\,c P<0.05 compared with negative control group, ns: non-significant.
X-ray and histopathological changes in joint showed highly significant anti- arthritic activity.
Table 10: Effect of Asparagus officinalis extracts on Arthritic Joints in X-Ray.
Groups (n = 6) mm

Group 1 (Normal Control, Tween 80 (l%v/v) + 0.5%w/v CMC in water (99%v/v))
Group 2 (Arthritic Control)
Group 3 (Methotrexate, 0.5 mg/kg)
Group 4 (Prednisolone, 5 mg/kg)
Group 5 (Asparagus officinalis, Petroleum ether, 75 mg/kg)
Group 6 (Asparagus officinalis, Petroleum ether, 150 mg/kg)
Group 7 (Asparagus officinalis, Petroleum ether, 300 mg/kg)
Group 8 (Asparagus officinalis, Ethanol, 75 mg/kg)
Group 9 (Asparagus officinalis, Ethanol, 150 mg/kg)
Group 10 (Asparagus officinalis, Ethanol, 300 mg/kg)
Group 11 (Asparagus officinalis, Water, 75 mg/kg)
Group 12 (Asparagus officinalis, Water, 150 mg/kg)
Group 13 (Asparagus officinalis, Water, 300 mg/kg) 6.29 ± 0.35 s (38.43)
10.21 ±0.64 8.32 ± 0.68 s (18.52) 8.29 ± 0.51s (18.86)
9.21 ± 0.44 c (9.87) 8.54 ± 0.57 s (16.38) 8.46 ± 0.20 s (17.15)
9.73 ± 0.42 (4.72) 8.83 ± 0.37 s (13.53) 8.34 ± 0.40 s (18.34)
9.38 ± 0.63 c (8.13) 8.32±0.31s(18.51) 7.93 ± 0.49 s (22.36)

F Value 23.75
P Value <0.0001
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test. The values are expressed as mean ± SD for each group (n=6), aJP<0.0001, hP<0.00\, c P<0.05 compared with negative control group, ns: non-significant.
The aqueous, ethanolic and petroleum ether extract were found to decrease RA factor, WBC's and Spleen weight and they are statistically significant. X-ray and histopathology were performed for joints and statistically significant efficacy was observed. Anti- inflammatory activity was performed using Paw edema model. Aqueous, alcoholic and petroleum ether extract has statistically significant anti- inflammatory effect from 2 hours to 6 hours at higher dose and less effect at lower doses also.
The results show that ethanolic and aqueous extract showing good anti- arthritic compared to petroleum ether extract against CFA induced arthritis of Asparagus officinalis also the aqueous, ethanolic and petroleum ether extract of this plant shows good Anti- arthritic and anti¬inflammatory activities but less analgesic activity.

We claim:

1. A formulation comprising the extracts of plant materials Asparagus officinalis,
Asparagus racemosus, Asparagus sprengeri, and Asparagus acutifolius thereof as an
active ingredient effective for analgesic, antiarthritis and antiinflammation for
preventing and treating arthritic diseases comprises therapeutically effective amounts
of extract of herb and petroleum ether or ethanolic or water in the following range by
weight: Asparagus officinalis - petroleum ether, 75-300 mg/kg, Asparagus officinalis -
ethanol, 75-300 mg/kg and Asparagus officinalis - water, 75-300 mg/kg;
wherein Asparagus officinalis is selected from the group consisting of Asparagus
racemosus, Asparagus sprengeri, and Asparagus acutifolius; wherein the formulation is in a form suitable for oral administration, wherein the form is selected from the group consisting of powder, capsule, tablet, liquid and
caplet.
2. A method for preparing the formulation as claimed in claim 1, comprising:
a. collecting the arial part of Asparagus officinalis and washing with water;
b. drying arial part of plant materials obtained from step a and making powder;
c. defatting the powdered drug obtained from step b with more than once with suitable
liquids to obtain extracts in Soxhlet apparatus;
d. drying extract under vacuum to result into solid form;
e. subjecting the extract to thin layer chromatography for separation of compounds; and
f conducting the study to determine the effects of the extracts paw edema, WBC's level
in blood serum and histopathological changes paw joints.
3. The method as claimed in claim 2, wherein the Asparagus officinalis extracting step
further comprises extracting the Asparagus officinalis residue with a petroleum ether to produce another extract.

4. The method as claimed in claim 2, wherein the Asparagus officinalis extracting step
further comprises extracting the Asparagus officinalis residue with an alcohol to produce another extract.
5. The method as claimed in claim 2, wherein the Asparagus officinalis extracting step
further comprises extracting the Asparagus officinalis residue with water to produce another extract.
6. The formulation as claimed in claim 2, wherein the formulation is conducted to evaluate
the analgesic activity and significant effect found at 180 min with aqueous, alcoholic and petroleum ether extracts.
7. The formulation as claimed in claim 1, wherein the formulation is conducted to evaluate
the anti- inflammatory effect 2-6 hours with aqueous, alcoholic and petroleum ether extracts.
8. The formulation as claimed in claim 1, wherein the formulation is conducted to evaluate
the anti- arthritic effect within day 14-21 with aqueous, alcoholic and petroleum ether extracts.