SCREENING OF INDIGENOUS HERBAL EXTRACTS FOR ANTI-INFLAMMATORY ACTION: IN VlTRO STUDIES USING RBC-MEMBRANE

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1 SCREENING OF INDIGENOUS HERBAL EXTRACTS FOR ANTI-INFLAMMATORY ACTION: IN VlTRO STUDIES USING RBC-MEMBRANE Introduction large number of anti-inflammatory drugs are in therapeutic use. Several others are undergoing clinical trial. The drugs include corticosteroids and their synthetic derivatives. non-steroidal anti-inflammatory drugs, gold salts, anti-rheumatic methotrexate. cyclasporine, etc. Most of these drugs are known to produce from mild to serious side effects. There is a growing demand for inexpensive and safe treatment methods. Inexpensive indigenous anti-inflammatory drugs are commonly used in indigenous systems of medicine. Active constituents such as phenolic compounds. flavonoids, coumarins. xanthones. lignans. triterpenoids, steroids, polysaccharides, alkaloids, etc. are reported to be responsible for the antiinflammatoty activity of the indigenous

2 Decrease in the stability of the erythrocyte membrane was noted in polyarthritis and in rheumatoid arthritis, due to changes in protein or lipoproteins of cellular membrane of erythrocytes.412 A wide variefy of drugs belonging to various pharmacological and chemical categories are known to protect erythrocytes against hypotonic hemolysis. Brown and later Bhaskar Rao ef and showed that non-steroidal anti-inflammatory drugs protected erythrocyte membranes from hypotonic hemolysis. The study of the erythrocyte membrane stabilization is simple and rapid, though non-specific. It is useful as a preliminary screening test for the potential anti-inflammatory compounds.418 In the present chapter, several synthetic non-steroidal anti- inflammatory drugs [beta-methasone sodium phosphate (Betnesol). diclofenac dium (Nac-50), oxyphenbutazone (Suganril), indomethacin (Microcid), piroxicam (Ddonex) and ibuprofen (Urufen-WO)] and indigenous herbal extracts [roots of Sida retusa L'-ln. (Malayalam: Kurumthotti) and Sida rhombifolia Linn. (Malayalam: Anakurumthotti), water of Cocm nucifem Lnn. (Coconut water), roots and leaves of Wex negrrldo Linn. (Malayalarn: Karinochi). Strobilan~es hepeanus Linn. (Malaydam: Karimkurinji) and IZcinus communis linn. (Malayalam: Avanakku), the whole plant of Phyffmthus nim~ Lnn. (Malayalam: Kizhikanelli) and Hydroco@/e asiatiw Lnn. (Malayalam: Kudakan), bulb of Alliurn cepa Linn. (Onion) and A//ium satiwrn Linn. (Garlic), rhizome of Curcurna longa Lnn. (Malaydam: Manjal) and leaves of Alstona xholan's Linn. (Malayalam: Pala) and Ocirnurn sanctum Linn. (Malayalam: Tulasi)] are screened for their anti-inflammatory action based on the obsewation that non-steroidal anti-inflammatory drugs protected erythrocyte membranes against hypotonic hemolysis.

3 111.2 Materials and Methods All of the chemicals used were of analflea1 reagent grade. The water used was double distilled with potassium permanganate. % drug extracts were prepared separately in water. For blood preservation, NIH Formula (i.e.. ACD) was used. NIH Formula A Trisodium citrate g. Citric acid. 8.0 g. Dextrose g. Distilled water ml. Used 80 ml. of NIH Formula A to preserve 500 ml. of blood. ACD blood was stored at 46 C in a refrigerator, avoiding frequent opening and dosing of the door, to prevent temperature elevation which would decrease the duration of safe storage. ml. of 0.2 M NaOH and 50 mi. of 0.2 M NaH2P04 were mixed and diluted to 1000 ml. with water to get 10 mm sodium phosphate buffer of ph = mm isotonic sodium chloride solution was prepared in 10 mm sodium phosphate buffer of ph = 7.4 (0.9% NaCl solution in 10 mm sodium phosphate buffer: ph = 7.4). A hypotonic saline solution (containing mm sodium chloride) was prepared in 10 mm sodium phosphate buffer of ph = 7.4 (0.5% NaCl solution in 10 mm sodium phosphate buffer: ph = 7.4). Erythrocyte lvsis in hypotonic solution was determined by assaying the release of hemoglobin following procedure of Seeman and ~einstein.~~~

4 Stock suspension of erythrocytes was prepared from the ACD preserved blood. Washed the erythrocytes thrice with the isotonic solution of NaCI. (154 mm sodium chloride in 10 mm sodium phosphate buffer: ph = 7.4). Prepared a suspension of the cells in isotonic buffered saline. Took different volumes (0.1 ml ml ml.. etc.) of the suspension and mixed with the pure buffer (Final volume = 5 ml.). Centrifuged for 5 minutes and read the absorbance of these ~olut!~)ns at 5 nm against water as the blank (zero absorbance). Selected the dilution giving a suitable absorbance for 100% hemolysis and selected a suitable volume of absorbance for 100% hemolysis. blood giving a suitable The experiments were carried out as follows: 1) To 0.1 ml. of stock erythrocyte suspension in a centrifuge tube. added 5 ml. of isotonic salt solution. incu'sated for 30 minutes at room temperature. centrifuged for 5 minutes and measured the ~Ssorbance at 5 nm as B :he absorbance of blank. 2) Absorbance corresponding to 100% hemolysis was determined as follows: To 0.1 ml. of stock erythr,te suspension in a centrifuge tube. added 4.5 ml. of sodium phosphate buffer. incubated for 30 minutes at room temperature, added 0.5 ml. of drug extract. cenhifuged for 5 minutes and measured the i.bsorbance at 5 nm as H. 31 To 0.1 ml. of stock erythrocyte suspension in a centrifuge tube. added 4 ml. of hypotonic solution and 0.5 ml. of water. incubated for 30 minutes at room temperature. added 0.5 ml. of drug extract. centrifuged for 5 minutes and measured the absorbance at 5 nm as C, the absorbance of control.

5 4) Absorbance of test (T) was found out as follows: To 0.1 ml. of stock erythrocyte suspension in a centrifuge tube, added 4 ml. of hypdonic salt solution and 0.5 ml. of drug extract, incubated for 30 minutes at room temperature, added 0.5 ml. of water, centrifuged for 5 minutes and measured the absorbance at 5 nm. The second. third and fourth steps were repeated for different drugs. C-B % hemolysis in control X = x 100 H-B T-B % hemolysis in test Y = x 100 H-B X-Y % inhibition by test - x 100 X Statistical Analysis The values reported are the average of the values from ten individual experiments in each Case k Standard Error of Mean 6.E.M). Statistical significance was estimated using Student's 't'-test Results and Discussion Lst of various synthetic non-steroidal anti-inflammatory drugs and indigenous herbal extracts screened for anti-inflammatory action and the results of screening have been summarized in Tables and

6 Table List of known (synthetic) drugs screened for mtl-inflammatory action and results Drug concentration - 1 tablet in 10 ml. water Amount of drug used for each experiment ml. Period of incubation - 30 minutes Temperature of incubation - Room temperature ph=74 (Values are the mean of 10 different experlnients in each case t S.E.M) r I I I No. Name of the drug hemlysis in I I I medium) ( control hemolysis in t-value inhibition 1 Beta-methasone sodium phosphate * 3.75 (Betnesol) lndomethacin (Microcid) / Ibuprofen (B~fen-0) k 1.87 a = p<0.01 b = 0.01<p<0.05 no symbol = not significant

7 Table Ust of indigenous herbal erttacte screened for anti-inflammatmy action and results Drug concentration - % in water ( g. in 100 ml. water) Amount of drug used for each experiment ml. (i.e g.) Period of incubation - 30 minutes Temperature of incubation - Room temperature ph = 7.4 (Values are the mean of 10 different experiments in St. No. 1 Name of tk drq Roots of Sida retusa Linn. each case i S.E.:.!) Dose (Weight in mg. ol crude drug in 1 mi. of incubatin medium) hemolysis in control i 3.61 / hernoiysis in test 38. i 1.18" di",',"",r,, conhvl and test group inhibition by test Water of Cocos nucifera Unn. undiluted water (0.5 ml.) t " Roots of Sida rhombifola Linn i i 2.27" Roots of Utex negundo Linn i " The whole plant of H'mtye asiatica Linn, t " BulbofNhumcepaLinn. 57.i k2.45 ' Leaues of Sfrobilanthes heyneanus Linn k %k1.86"

8 ~ - 32t Contd... Table Si. No. Name of the drug Dose (Weight in rng. of crude drug in 1 mi of incubation medium) hemohjsis in control hemolysis ~n test t-value behwen control and test pups inhibition by test Rhizome of Curcuma longa Linn. Bulb of Allium satiwm Linn. Leaves of.alstonia scholaris Linn. Roots of.strobilanthes heyneanus Lnn. I ' i i * r 3.39" t 1.22" t 2.78" Rook of Rfcinu communis Linn i t 1.55" Leaves of Utex negundo Linn " The whole plant of Phyllanthus niruri Linn i_ " Leaves of Ricinus communis Linn ~ Leaves of Ooinom sanctum Linn. a = pco.01 b = 0.01cpc0.05 no symbol = not significant t

9 cine data presented in Table m.1 show that. of the six clinically important non-steroidal anti-inflammatory drugs screened, all except indomethaan, piroxicam and ibuprofen, significantly stabilized erythrocyte membrane against hypotonic hemolysis. Indomethacin and piroxicam also stabilized the erythrocyte membrane against hypotonic hemolysis but the effect was less significant. The effect of ibuprofen was insignificant. The stabiling effect of the non-steroidal anti-inflammatory drugs on erythrocytes may be due to a stabilizing effed of the drugs on certain proteins in the cell membranes. The association of these drugs with biological membrane of cells and cell organelles is assumed to produce a change in the selective permeability of the membrane, which has profound biochemical implications. The data pre;ented in Tables and 111.2, indicate that all the indigenous herbal extracts screened, except leaves of &nus cornrnunis Linn. and Ocimum sanctum Iinn., offered significant protection to the erythrocyte membrane and are comparable to the synthetic non-steroidal antiinflammatory drugs in protecting the erythrocyte membranes from hypotonic hemolysis. The indigenous drug : leaves of &nus cornrnunis Lnn. stabilized the erythrocyte membrane against hypotonic hemolysis but the effed was found to be significant only to a lesser extent. The effect of leaves of Ocimum sanctum Linn. was insignificant. Among the synthetic drugs tested, beta-methasone sodium phosphate was found to be the most potent anti-inflammatory drug. Among the herbal extracts screened, the aqueous extmd of roots of Sida retusa Linn. exhibited the most potent anti-inflammatory action. It can be inferred from the data presented in the Tables and that most of the indigenous herbal extracts tested exhibited significant anti-inflammatory action comparable to the drugs employed in current clinical practice.