A Preliminary Study on In Vitro Antioxidant and In Vivo Anti-Inflammatory Activity of Cissus quadrangularis Linn.
Mamta Tiwari1*, Pushpraj S Gupta2, Nisha Sharma3
1Research Scholar, Christian School of Pharmacy, SHUATS, Prayagraj (U.P.)
2Associate Professor, Christian School of Pharmacy, SHUATS, Prayagraj (U.P.)
3Associate Professor, University Institute of Pharmacy, C.S.J.M.U, Kanpur.
*Corresponding Author E-mail: mamta1712@gmail.com
ABSTRACT:
Cissus quadrangularis L. is a member of succulent climber of family Vitaceae and it is also called as bone setter because of its bone healing property. The present study focused to assess the in vitro antioxidant and in vivo anti-inflammatory activity of the plant stem extracts in different solvents namely petroleum ether, chloroform, ethyl acetate and methanol. Among all these extracts; ethyl extract showed highest phenolic and flavonoid content (19.35±0.29mg GA/g, 8.68±0.06 RE/g dry wt. respectively). In DPPH free radical scavenging assay all mentioned extracts exhibited free radical scavenging activity but the highest free radical scavenging activity was showed by ethyl acetate extract in increasing dose dependent manner. On the basis of highest phenolic, flavonoid content and free radical scavenging activity ethyl acetate extract was selected for anti- inflammatory activity. The anti-inflammatory activity of Cissus quadrangularis Lin. was investigated in carageenan induced inflammation in Wistar albino rats. Two different doses 250mg/kg and 500mg/kg were administered to the animals. The results showed that both doses had significant reduction in inflammatory condition but dose 500mg/kg showed maximum inflammatory reduction on comparing with control and standard treated groups.
KEYWORDS: Cissus quadrangularis, Anti-inflammatory, Antioxidant, ethyl acetate, Flavonoids, Free radicals.
INTRODUCTION:
Inflammation is a biphasic response, the initial response (1-2 hours) which is mainly initiated by mediators like histamines, serotonin and increased production of prostaglandins in the injured area and late response is maintained by prostaglandin release and mediated by bradykinin leukotrienes. Inflammation is characterised by vasodilatation which is due to increased blood flow causing the redness (rubor), increased heat (color), enhanced permeation capability of the blood vessel leads to leakage (exudation) which induce swelling, tumor in the affected area4. Mediators like Bradykinin increase reactivity to pain (hyperalgesia, dolor)1.
In present scenario, Non-steroidal anti-inflammatory drugs or steroidal agents are the very frequently prescribed drugs for treating many inflammatory conditions. These agents are not free of side effects5, due to this reason now a day’s people are switching towards drugs of plant or natural origin. More and more scientific research are going on to develop pain-killer and anti-inflammatory agent of natural origin because of their effectiveness, very low incidence of side effects and provide a pathway to discover some more valuable synthetic molecules. Inspite of these, plant originated compounds are cheaper than synthetic preparations existing in the Pharmaceutical market6.
According to World Health Organisation WHO more than 80 percent of the world’s population trust on traditional medicines for their primary health issues7. More than 40 percent current medicines contain ingredients that are derived from plants8. Traditional medicines of plant origin contain various active secondary metabolites like alkaloids, steroids, terpenoids, polyphenols etc. which can be beneficial in different types of disorders. Now it is a demand to discover anti-inflammatory drugs of plant origin with minimum unwanted reactions and greater effectiveness6.
Cissus quadrangularis:
Cissus quadrangularis L, belongs to family Vitaceae, is a common succulent perennial climber, characterized by a thick quadrangular fleshy stem, is an edible plant, can be grown in all parts of India, especially in tropical regions9. This plant has ability to join the bones and due to this property this plant is also called as bone setter. This plant is called by various other names in different languages in different parts of the country like Nalleru, Vajravalli, Hadjod, Kandvel, Hadbhanga, Vedhari, Perandai, and Veldgrap and Edible-stemmed vine in English. It is also known as Vitis quadrangularis10.
Description of the plant:
Cissus quadrangularis is a herb, reaching a height of 1.5m. It grows almost in all parts of India but particularly in hotter region and it can be cultivated by cutting of stem in month of June-July11. Stem is quadrangular with internodes 4-5 cm in length and 1 to 2 cm wide. The surface is fibrous, smooth, glabrous, buff colored with greenish tinge, dichotomously branched, angular portion reddish-brown, no taste and odour12.
Tendrils are simple, stems become leafless when old. Leaves are simple 2.5-5cm long, reniform or ovate broadly, sometimes lobed, dentate, glabrous. Fleshy green stem contributes a great portion of aerial parts while only a small portion, 5- 8% is contributed by the leaf part of the aerial plant parts13. Small greenish white flowers appears in short umbellate cyme, Fruit is fleshy berries, obovoid or, succulent11,12.
MATERIALS AND METHODS:
Collection and authentication:
Cissus quadrangularis was collected from the surrounding area of Indira Nagar, near C.S.J.M. University, Kanpur. The plant botanically identified and authenticated by Botanical Survey of India, Dehradun with authentication no. BSI/NRC Tech/Herb (ident.)/ 2015-16/301.
Preparation of plant extract:
The plant material of Cissus quadrangularis was washed, cut into small pieces, air-dried and powered with the mechanical grinder. The required amount of powder was extracted in a Soxhlet extractor, with solvents of increasing polarity petroleum ether, chloroform, ethyl acetate and methanol using sequential extraction. After this, with the help of rotary evaporator (Evator Mumbai), under reduced pressure the obtained extracts were dried and its percent yield was determined. The solid extracts were kept in air tight container and stored in refrigerator for future use.
Determination of total phenolic content:
The total phenolic content of the various extracts (petroleum ether, chloroform, ethyl acetate and methanol) of Cissus quadrangularis was determined by Folin-ciocalteu method by using U. V. Spectrophotometer and Gallic acid as a standard. In dry and clean test tubes, 0.1 ml of extracts were combined with 0.5ml of Folin-Ciocalteu reagent and diluted with distilled water and volume make up was done upto 3 ml and mixed thoroughly. After incubating this mixture for 5 minutes at room temperature, 2 ml sodium carbonate (Na2CO3) was added to this content and mixed thoroughly. This mixture was further incubated for 60 minutes at cool dark place. The absorbance of the mixture was read at 650 nm with the help of U.V. Spectrophotometer. All determinants were done in triplicate for all extracts14.
Determination of total flavonoid content:
The total flavonoid content of the various extracts (petroleum ether, chloroform, ethyl acetate and methanol of Cissus quadrangularis was determined by aluminium chloride method15 and rutin was taken as standard. According to this method, in clean and dry test tubes containing 0.1ml of extract sample, added 0.5ml of aluminium chloride, and volume was made upto 3ml with distilled water. After 5 minutes of incubation, 2ml of sodium hydroxide solution was added to test tubes and shaken thoroughly. The resulting mixtures were incubated for 5 minutes at 50°C and cooled at room temperature. The absorbance of mixture was read at 420 nm. All experiments were performed in triplicate. The total flavonoid content was evaluated and the result was exhibited as mg rutin per g dry weight16.
Determination of free radical activity by DPPH method:
The Principle of DPPH (1,1-diphenyl-2-picrylhydrazyl) antioxidant assay was used to determine the potential of substance to scavenge the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). Antioxidant compound which releases an electron or hydrogen atom that is accepted by DPPH molecule due to which DPPH is converted to a more stable DPPH molecule. This process converts DPPH in its reduced form which gives pale yellow colour. If an antioxidant compound is having greater or higher free radical reactive oxygen molecules scavenging property then this capacity may reduce more of DPPH molecules and sample would be less purple in colour17.
Free radical scavenging activity of various extracts was assessed by DPPH method. Different concentrations (50, 75, 100 and 125 µg/ml) of extracts of Cissus quadrangularis were transferred in clean and dried test tubes and volumes were adjusted to 2ml with acetate buffer, pH 5.5. After this 0.03mM solution of DPPH prepared in methanol and 500µl of this solution was added to 1ml of the extract at different concentrations13. The reaction mixture were mixed thoroughly and for 30 minutes, kept in the dark at room temperature. The absorbance of mixture was determined at 517nm by using double beam U.V. spectrophotometer. The absorbance was measured against blank. The butylated hydroxyl anisole (BHA, 1mg/ml) was used as the standard18.
Acute oral toxicity study:
various research studies suggested that Cissus quadrangularis is well tolerated at higher doses. In an acute toxicity study, hydroalcoholic extract of Cissus quadrangularis stems at a dose of 500, 1000, 1500, 2500, 3000, 3500, 4000, 4500 or 5000mg/kg body weight were given orally to the rats. The rats were continuously observed for 2 hr then frequently upto 6 hrs and then onwards for 30 days. Results of this study suggested that Cissus quadrangularis was safe even at higher dose of 5000mg/kg body weight as this dose did not exhibited any mortality in rats within 30 days of experiment19.
Experimental Animals:
Wistar albino rats (150-210g) of both sex were allowed in the study. The animals were kept under standard laboratory conditions. Animals were kept according to ethical guidelines under standard laboratory conditions, at room temperature 25-30°C with relative humidity 60 to 65% and 12 hours light and dark cycle. Animals were fed with standard laboratory diet and water ad libitum. The experimental protocol was approved by Institutional Animal Ethics Committee and the approval no. was IAEC/SHIATS/PA16III/SMTPG02. The experiment protocol was performed according to guidelines of Committee for the Purpose of control and Supervision of Experiment on Animals.
Anti-inflammatory activity study:
Carageenan induced paw edema in rats:
The anti-inflammatory activity of the ethyl acetate extract was assessed by using carageenan induced paw edema. Adult Wistar rats of either sex (150-200g) were divided in four groups and each group was having 6 animals20.
Group I: Normal control (0.5ml distilled water) p.o
Group II: Standard drug (diclofenac sodium 10mg/kg) i.p.
Group III: Ethyl acetate extract of Cissus quadrangularis (250mg/kg)p.o
Group IV: Ethyl acetate acetate extract of Cissus quadrangularis (500mg/kg) p.o
After 30 minutes of administration of drugs and different doses of ethyl acetate extract, 0.1ml of 1% carageenan in normal saline was injected into subplantor region of the left hind paw of each rat to induce edema. The differences volume of paw edema was calculated at intervals of 0, 1, 2, 3, 4 and 5 hrs after carageenan injection with the help of Digital plethysmometer (Medicaid, Chandigarh). The percentage inhibition was calculated of by using following formula21.
Percentage inhibition= [Vc-Vt/Vc] × 100
Vc - Volume of paw edema in control group
Vt – Volume of paw edema in treated group
Statistical Analysis:
The statistical analysis was carried out using PRISM software version: 4. The data for paw edema volume is expressed as mean ± SD of 6 observations. All data was analyzed by two way ANOVA followed by Benferroni post tests to calculate inter difference between groups. Statistical significant was ***P<0.001, **P<0.01, *P<0.05 not significant when compared to control and other groups.
RESULTS AND DISCUSSIONS:
Total Phenolic Content (TPC) of methanol, petroleum ether, ethyl acetate and chloroform extract is shown in Figure 1. Total phenolic content of Petroleum ether, Chloroform, Ethyl acetate and Methanol extract were 0.4 ±0.2, 2.93±0.85, 7.66±1.106, 4.6±0.632 gallic acid equivalent(GAE) gram of dry extract respectively. Among different extracts, ethyl acetate extract of CQ was having highest phenolic content. The phenolic content was calculated according to following equation from the standard as gallic acid graph Figure 2.
Absorbance = 0.001 (GA) μg + 0.647(R2 =0.991)
Figure1. Bar Diagram Showing Total Phenolic Content for Various Extracts of Cissus Quadrangularis.
Figure 2: Gallic acid standard calibration curve.
Total Flavonoid Content (TFC) of methanol, petroleum, ethyl acetate and chloroform extract is presented in Figure 3. TFC of Petroleum ether, Chloroform, Ethyl acetate and Methanol extract were 0.3 ±0.251, 3.6 ± 0.624,19.76 ±1.59, 10.57±0.589 as mg rutin per g dry weight respectively. Highest flavonoid content was found in ethyl acetate extract as compared to other extracts. The total flavonoid content result was calculated according to following equation from the standard rutin (Figure 4).
Absorbance = 0.003 rutin (μg) + 0.154 (R2 0.978)
Figure 3: Bar diagram showing total flavonoid content in various extract of Cissus quadrangularis L.
Figure 4: Rutin standard calibration curve.
According to various research studies stem part of Cissus quadrangularis enriched with various important constituents like phenolics compounds, tannins, Vitamin C, Carotenoids, which are known for antioxidant property22. Free radicals are highly toxic species which are produced by different biochemical and photochemical reactions23. These phytoconstituents might be responsible for its high in vitro DPPH antioxidant activity that was expressed by ethyl acetate extract. Various researchers suggest that flavonoids and phenolic phytoconstituents are associated with treating various disorders.
All four types of extracts had exhibited free radical scavenging property in dose dependent manner at different concentrations but ethyl acetate extract showed highest antioxidant activity than other extracts. This extract had scavenged 88 % of DPPH radicals at concentration 125 μg/ml which was near about the standard BHA that is 93%, methanol 65%, chloroform 61% and pet ether 37% scavenged at same concentration Figure 5.
Figure 5: DPPH radical scavenging activity of various extracts of Cissus quadrangularis.
The experimental study was conducted to evaluate anti-inflammatory property of most potent ethyl acetate extract of Cissus quadrangularis in carrageen induced paw edema animal model. Carageenan-induced paw edema is mostly preferred method to investigate the level of acute inflammation and effectiveness of various doses of tested drugs. Acute inflammation involves two phase response. In the first phase (1-2 hrs), there is release of different types of chemical substances such as histamine and serotonin along with synthesis of prostaglandins around the area of damaged tissues24. The second phase (3-5hrs) is produced by release of kinins mainly prostaglandins proteases and lysosomes25.
In present study there was a slow elevation in the paw volume in control animals group. Diclofenac sodium is a potent nonsteroidal anti-inflammatory drug, with dose 10mg/kg, it inhibited progression of paw edema significantly from 1st hr onwards. It exhibited maximum reduction (68.42%) at the end of 5th hr. Although both the doses (250 and 500mg/kg body wt) of ethyl acetate extract of Cissus quadrangularis were effective in reducing carageenan induced paw edema significantly from the first hour of carageenan administration in a time dependent manner but maximum percentage inhibition was observed with highest dose (500mg/kg body wt) at the 5th hr which was almost nearer to standard drug. There was 58.63% and 64.79% reduction in paw edema with dose 250 and 500 mg/kg body wt of EACQ respectively at the 5th hr of second phase Table 1 and Figure 6 and 7.
Table 1. Effect of Ethyl Acetate extract of Cissus quadrangularis L. in Carageenan induced edema.
|
Group |
Treatment (mg/kg) |
0 hr |
1 hr |
2 hrs |
3 hrs |
4 hrs |
5 hrs |
|
I |
Distilled water 0.5 ml/rat,p.o |
0.67 ±0. 095 |
1.30 ± 0.0195 |
1.68 ±0.0094 |
1.77 ±0.0134 |
1.90 ±0.0121 |
1.96 ±0.0138 |
|
II |
Diclofenac sodium 10mg/kg bw,i.p |
0.64 ±0.0134 |
1.04 ±0.0068*** |
1.38 ±0.0068*** |
1.24 ±0.0106*** |
0.81 ±0.0106*** |
0.60 ±0.0129*** |
|
III |
CqEA 250mg/kg bw,p.o |
0.65 ±0.0194 |
1.15 ±0.0074** |
1.56 ±0.0125*** |
1.49 ±0.0074*** |
1.23 ±0.0110*** |
0.81 ±0.0106*** |
|
IV |
CqEA 500mg/kg bw,p.o |
0.65 ±0.0149 |
1.10* ±0.0146 |
1.49 ±0.0115*** |
1.33 ±0.0068*** |
1.03 ±0.005*** |
0.69 ± 0.0074*** |
Mean paw volume in ml at different hrs of carageenan injection Each value represents the mean ± SD, n=6 in each group, obtained data was analyzed by using two way ANOVA followed by Bonferroni post tests to compare the values, where statistically significance was ***P<0.001,** P<0.01 and *P<0.05 not significant.
|
|
|
|
Figure 6. anti-inflammatory property effect of different doses of ethyl acetate extract of Cissus quadrangulais L. on Carageenan-induced paw edema at different time intervals.
|
Figure 7. Bar diagram showing percentage inhibition of paw edema by ethyl acetate extract of Cissus quadrangulais L. at different time intervals.
|
CONCLUSION:
Wound is one of the prominent causes for malformation and death and thorough investigation in this field provides different types of healing agents26. Analysis of experimental data concluded that ethyl acetate extract of Cissus quadrangularis had comparable anti-inflammatory activity. At dose 500 mg/kg body wt, it showed effect which was significant to standard drug. This justifies the use of this plant for various disorders since a long time. Phytochemical analysis of extract indicated presence of different chemical constituents which may be associated with anti-inflammatory activity. Hence the study concludes that flavonoids and phenolic component may potentiate the activity of extract. Further studies are required for the detection of active chemical constituents and their mechanism of actions.
ACKNOWLEDGEMENTS:
Corresponding author is very grateful to Dr. C. S. Bhargava, Director, Advance Institute of Biotech and Paramedical Sciences, Kanpur for providing necessary facilities to carry out this work.
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Received on 12.04.2020 Modified on 09.06.2020
Accepted on 01.08.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(5):2619-2624.
DOI: 10.52711/0974-360X.2021.00461