Activity of Plumbago zeylanica Linn. root and Holoptelea integrifolia Roxb. bark pastes in acute and chronic paw inflammation in Wistar rat.

BACKGROUND: The pastes prepared from roots of Plumbago zeylanica Linn. and barks of Holoptelea integrifolia Roxb. are widely used by traditional healers for the treatment of arthritis in rural northern Karnataka.
OBJECTIVE: The present study was undertaken to scientifically evaluate the safety and efficacy of traditionally used formulations in experimental animals.
MATERIALS AND METHODS: The study, approved by IAEC was carried out in male Wistar rats and dermal toxicity in rabbits. Carrageenan model was used to assess effect on acute inflammation. Paw volume were measured at 1, 2, 4, and 6(th) hour postchallenge. Chronic inflammation was developed by using Complete Freund's Adjuvant (CFA). Paw volume, ankle joint circumference, and body weight were assessed on 1(st), 4(th), 8(th), 14(th), 17(th), and 21(st) day. Paste was applied once every day to the inflamed area of the paw of respective groups of animals, continuously for 14 days. STATISTICS: The data were analyzed by one way analysis of variance followed by Dunnett's post hoc test. P ≤ 0.05 was considered as significant.
RESULTS: The formulations did not show any dermal toxicity and found to be safe. Both the pastes significantly (P < 0.05) suppressed, carrageenan-induced paw edema at 6(th) hour and Holoptelea integrifolia appears to be more effective than Plumbago zeylanica. Significant reduction was observed in paw volume, ankle joint circumference and animal body weight gained.
CONCLUSIONS: The tested formulations (P. zeylanica root and H. integrifolia bark pastes) showed significant antiinflammatory activity. The present findings therefore support its utility in arthritic pain, inflammation and the claim of traditional practitioners.

INTRODUCTION

Rheumatoid arthritis (RA) is a symmetric, polyarticular, progressive disease that primarily affects small diarthodial joints of the body.[1] It is most common form of arthritis and has a substantial societal effect in term of cost, disability, lost productivity[2] and is estimated to affect 0.5-1% of the population worldwide. It afflicts people of all races equally and can occur at any age but most often starts at the age of 40-70 years. Patients seeking relief are subjected to treatment with nonsteroidal antiinflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), or immunosuppressant drugs, that provide prompt remissions at the cost of varieties of adverse drug reactions, which often force to discontinue the therapy. Most of the patients who believe that alternate systems of medicine are safer and effective usually opt for herbal medicine. Apart from qualified Ayurvedic practitioners, many traditional healers dispense a variety of herbal preparations for common ailments. In rural area of Belgaum district Plumbago zeylanica and Holoptelea integrifolia locally known as Chilbil/Kanju/Papri preparations are routinely dispensed for topical application to treat arthritis. Plumbago zeylanica Linn. belongs to the family Plumbaginaceae, which is a perennial, rambling undershrub, much cultivated and readily spread throughout India.[3] The antiinflammatory activities of P. zeylanica extracts, administered orally, have been reported in animal models of acute inflammation.[45] Holoptelea Integrifolia Roxb. belongs to the family Ulmaceae, which is large sized tree, found all over India, tropical and subtropical regions of Asia and Africa.[6] The aqueous leaf extract of H. integrifolia is reported to possess antiinflammatory activity.[7] However, there are no reports regarding their efficacy by topical application against inflammation as being used by traditional practitioners for the treatment of arthritis. Their efficacy has not been evaluated scientifically in chronic model of arthritis by topical application. Therefore, the present study was undertaken to explore their antiinflammatory activity by topical application to resembles RA in carrageen inflammation and Complete Freund's adjuvant (CFA)-induced arthritis in Wistar rats.

MATERIALS AND METHODS

Documentation of the traditional practice

The selected traditional practitioners were frequently visited and the details on disease, diagnosis, treatment, formulation and its ingredients, method of preparation, mode of administration, etc., were documented through interview and discussions.

Plant material

The plants incorporated in the formulations, that is, Plumbago zeylanica L. (RMRC-524) and Holoptelea Integrifolia Roxb. (RMRC-525) were collected from the local forest areas of Khanapur and Jamboti. Plants were authenticated and their voucher specimens were deposited at the herbaria of Regional Medical Research Centre (ICMR), Belgaum.

Preparation of paste

The formulations were prepared by following the exact procedures of traditional practitioners.

Five grams of dried root of P. zeylanica was weighed and 25 ml of distilled water was measured. The root piece of P. zeylanica was rubbed over a clean stone surface for 15 min by adding 5 ml of distilled water in five installments. The resulting paste was Weighed and consistency was semisolid paste. The P. zeylanica root piece was weighed again to find out the weight loss due to rubbing. The process was repeated for six times, to find out statistical mean. Appearance of paste was Brownish [Figure 1], viscosity was (1333 cps) measured by Brookfield viscometer and pH was 6.3. This Paste (PZRP) was applied in sufficient quantity on the surface of the inflamed area and warmed with radiant heat (100 W electrical bulb placed at a distance of 20 cm for 15 min) to mimic the technique as practiced by traditional healers.

Figure 1

Pastes of Plumbago zeylanica roots (PZRP) and Holoptelea integrifolia bark (HIBP)

Bark of H. integrifolia was rubbed with friction on a clean stone surface with adequate quantity (25 ml distilled water) of distilled water to obtain a thick paste [Figure 1]. Appearance of paste was brownish, viscosity was 1285 cps, and pH was 6.6. This paste (HIBP) was applied in sufficient quantity on the surface of the inflamed area, wrapped with wet cotton gauge and was covered with medical bandage (simulation of technique as used in traditional practice).

Animals

Healthy, adult Wistar rats of either sex weighing 150-200 g and New Zealand white rabbits weighing 1.5-2.0 kg were procured from Shree Venkteshwara Traders, Bangalore, India. They were housed in the laboratory for about a week for acclimatization at room temperature (25 ± 3°C) with natural light and dark cycle. Rats were fed with standard rat chow, tap water ad libitum, whereas rabbits, housed in cages, were fed with balanced diet formulated in the department (cauliflower, cabbage, Bengal gram, and wheat flour) and tap water ad libitum. The experiments were conducted subsequent to the approval and permission from IAEC, constituted as per CPCSEA guidelines.

Drugs and chemicals

CFA, carrageenan, and diclofenac gel were purchased from SIGMA Chemicals co (St Louis, MO). Normal saline was purchased from Claris life science Ltd and diclofenac diethylamine gel ‘INAC gel’® : (Zydus Alidac) was purchased from local market.

Preliminary phytochemical screening

The paste was screened for the presence of various phytoconstituents, namely, alkaloid, steroids, flavonoids, glycoside, protein, carbohydrate, and phenolics.[89]

Acute dermal toxicity studies

Acute dermal irritation test of traditionally used pastes of P. zeylanica and bark of H. integrifolia were carried out in individually caged healthy New Zealand rabbits, weighing 1.5-2.0 kg. The fur around both the flanks in an area measuring 6 × 6 cm2 was clipped close to the skin without injuring the skin. After 24 hours of clipping, the preparation was applied randomly on one side and the vehicle on the other and the observation were carried out as per Organization for Economic Cooperation and Development (OECD) 404 guideline 2002.[10]

Antiinflammatory activity

Male Wistar rats with a body weight between 150 and 200 g were starved overnight, with free access to water. First group (control group) received normal saline, second group (standard group) received diclofenac diethyl amine gel, third and forth group (test groups) received PZRP and HIBP, respectively. All were topical application to the paw of respective animal groups. After 30 min of paste application, the rats were challenged by a subcutaneous injection of 0.05 ml of 1% solution of carrageenan into the plantar region of the hind paw.[11] The paw was marked with ink at the level of lateral malleolus to facilitate uniform dipping in mercury to measure the paw volume. The paw volume was measured plethysmographically immediately after injection, and 1, 2, 4, and 6th hour postchallenge of carrageenan. The increase in paw volume has been expressed as percentage increase over control recorded in terms of milliliter (ml). Percent inhibition of inflammation was calculated using the formula:

where Vc and Vt represents average paw volume in control and treated group, respectively.

Antiarthritic activity

Adult healthy male Wistar rats with a body weight between 150 and 200 g were used. On day 1, animals were injected intradermally into the sub plantar region of the right hind paw with 0.1 ml of CFA.[12] Application of the paste or the standard was started on the same day and continued for 14 days as practiced in traditional practice. Paw volumes, ankle joint circumference and body weight were recorded on the day of injection. The severity of the induced adjuvant disease was assessed by observation of the injected paw. The paw volumes (ml), paw thickness (mm), ankle joint circumference (cm), and body weight (g) were assessed on day 1st, 4th, 8th, 14th, 17th, and 21st.

Statistical analysis

Data were expressed as Mean ± SEM and were analyzed by one way analysis of variance (ANOVA) followed by Dunnett's post hoc test and P ≤ 0.05 was considered as significant.

RESULTS

PZRP was found to be positive for the presence of carbohydrate, glycoside, steroids, alkaloids, and flavonoids.

HIBP was found to be positive for the presence of carbohydrate, glycoside, alkaloid, phenolics, steroids, and flavonoids.

There were no signs of toxicity and preparation on single application appears to be devoid of major dermal irritation.

The severity of carrageenan-induced inflammation was significantly decreased in PZRP (P < 0.05) and HIBP-treated groups (P < 0.01) as compared with that of control group and were comparable to diclofenac gel-treated group [Figure 2].

Figure 2

Antiinflammatory activity of P. zeylanica root and H. integrifolia bark pastes N = 6, *= P≤0.05, **=P≤0.01

The severity of CFA-induced inflammation was significantly suppressed by the treatment with PZRP (P < 0.05) and HIBP (P < 0.01) as compared with that of control group and were almost comparable to that of diclofenac gel-treated group. Paw volume was measured in milliliter (ml) [Figure 3], ankle joint circumference was measured in centimeter (cm) [Figure 4], and body weight changes in grams (g) [Figures 5].

Figure 3

Paw volume changes in various treated groups of chronic inflammation n = 6, a = P < 0.05, b = P < 0.01

Figure 4

Ankle joint circumference changes in various treated groups of chronic inflammation n = 6, a = P < 0.05, b = P < 0.01

Figure 5

Body weight changes in various treated groups of chronic inflammation n = 6, a = P < 0.05, b = P < 0.01

DISCUSSIONS

Even though numbers of antiarthritic agents are available for the treatment of arthritic disorders such as Adalimumab, Infliximab, Abatacept, Rituximab and Aurothiomalate they are neither safe nor affordable for common/poor people. Most of them share common adverse effect like, upper respiratory tract infections, urinary tract infections, dermatitis, stomatitis, transient hematuria, mild proteinuria etc. In the present study PZRP showed 4%, 12%, 9% and 32% suppression in carrageenan model of inflammation at 1st, 2nd, 4th and 6th hours, respectively, which is insignificant compared with the control group. The standard, diclofenac diethyl amine treated group has showed 10%, 42%, 69% and 75% suppression at 1st, 2nd, 4th and 6th hour, respectively, in which suppression at 4th and 6th hour is significant with a value of P < 0.01. The protection in HIBP-treated group was almost comparable with that of diclofenac diethyl amine. HIBP has showed 37%, 25%, 45% and 64% suppression a 1st, 2nd, 4th and 6th hour, respectively, in comparison to control group, of which suppression at 6th hour is significant (P < 0.01). In CFA-induced chronic inflammation model, paw volume and ankle joint circumference are significantly suppressed in both treated groups, which are comparable to that of diclofenac gel. Paw volume was significantly suppressed on 4th and 8th day in HIBP, PZRP and diclofenac get-treated group with a significant value (P < 0.05). While on 14th, 17th and 21st day, HIBP, PZRP and diclofenac gel treated group showed significant suppression with P < 0.01. Ankle joint circumference was progressively increased in control group, whereas in HIBP, PZRP and diclofenac gel treated group showed significant suppression with a value (P < 0.01) on 17th and 21st day. On 8th and 14th day, diclofenac gel, HIBP and PZRP showed significant (P < 0.01 and P < 0.05) suppression, respectively. Body weight was drastically declined in the control group, whereas in HIBP, PZRP and diclofenac gel-treated group, weight gain was shown on 8th and 14th, 17th and 21st day (P < 0.05 and P < 0.01), respectively. There is paucity of information regarding HIBP and PZRP safety and efficacy in joint pain disorder. The reported antiinflammatory activity of P. zeylanica and H. integrifolia supports its antiarthritic potential. However, both plants have been reported to have β-sitosterol as one of its active constituents, which is known to be steroid. The antiarthritic effect could be contributed due to the β-sitosterol, flavonoids, and alkaloids. It is desirable to elucidate the antiarthritic mechanism of the used plants, prior to establish its efficacy in large number of patients suffering from joint pain disorders. Therefore, we are further studying the chemical moieties responsible for antiinflammatory activities and elucidate their mechanism of action.

CONCLUSION

Based on the gathered data, we can conclude that, PZRP and HIBP are effective against CFA-induced arthritis model of Wistar rats.