DOI: 10.14457/tu.the.2014.660
title: Anti-inflammatory and antioxidant activities of Thai traditional remedy extract called “Leard-ngam” and its plant ingredients

description: Leard-ngam remedy (LG) as a Thai traditional remedy has long been used to treat primary dysmenorrhea including pain and inflammation. It is in the Thai National List of Herbal Medicines Product A.D.2011 and also used in hospitals and medical clinics, Thailand. Its formula consists of 20 herbal ingredients. The objectives of the study were to investigate anti-inflammatory and antioxidant activities of the extract of LG remedy and its plant ingredients. The active compounds for markers of LG remedy also isolated by chromatographic techniques. The stability at accelerated condition of LG remedy extract was also tested by biological activity.LG remedy was extracted similarly to that practiced by Thai traditional doctors (ethanol and aqueous extraction). The yield of ethanolic extracts of LG and it herb components were in range from 17.60% to 0.77% and the yield of aqueous extracts ranged from 62.24% to 3.22%. The ethanolic extract of Citrus hystrix DC. (17.60%) and the aqueous extract of Allium sativum Linn. (62.24%) showed the maximum yield. The anti-inflammatory activity was tested as their inhibition of NO and PGE2 production. The results for inhibition of NO production found that five extracts from forty three extracts showed inhibition of NO production more than 70% (IC50 less than 30 µg/ml) and non-cytotoxicity. Four ethanolic extracts were Zingiber officinale Roscoe., Zingiber cassumunar Roxb., Oroxylum indicum (Linn.) Kurz. and Piper sarmentosum Roxb. (IC50 of 2.87±0.31, 4.93±0.42, 5.64±0.45, 10.96±0.65 µg/ml, respectively). Aqueous extract of Syzygium aromaticum Linn. Merr. Et Perry. exhibited anti-inflammatory activity (IC50 of 21.00±0.57 µg/ml). The 95% ethanolic extract of LG remedy had non-cytotoxicity and moderated anti-inflammation activity with IC50 of 28.18±4.63 µg/ml. In the positive control, Indomethacin showed IC50 of 25.04±3.79 µg/ml. The most effective extracts with NO inhibition activities were selected to tested inhibitory activity against LPS induced PGE2 production. It was found that four ethanolic extracts of Piper nigrum Linn., Zingiber officinale Roscoe., Zingiber cassumunar Roxb., Oroxylum indicum (Linn.) Kurz., and Leard-ngam remedy exhibited high anti-inflammatory activity via inhibition of PGE2 production, of which IC50 values equal to 1.20±0.05, 4.78±1.60, 7.45±0.01, 8.17±0.15 and 8.59±0.89 µg/ml, respectively. As the standard control, IC50 value of Indomethacin is 1±1.02 µg/ml).The antioxidant activity was determined by DPPH radical scavenging assay. This result showed that ethanolic extracts of Syzygium aromaticum Linn.Merr. Et Perry, Oroxylum indicum (Linn.) Kurz., Zingiber officinale Roscoe. showed high antioxidant activity, with EC50 values of 9.20 ± 0.29, 9.94 ± 0.91 and 14.34 ± 0.28 µg/ml, respectively. Two aqueous extracts of Syzygium aromaticum Linn.Merr. Et Perry. and Amomum xanthioides Wall. show high antioxidant activity, with EC50 values of 5.83±0.17, 11.91±0.83, respectively, whereas the standard BHT showed mean EC50 value of 15.84±1.42 µg/ml. The most effective LG extract with biological activities, the ethanolic extract of LG (LG1) was separated using vacuum liquid chromatography (VLC), yielding 5 fractions of LGa, LGb, LGc, LGd, and LGe. The fractions of LG extract were studied for anti-inflammatory by NO inhibitory assay and antioxidant activities by DPPH scavenging assay repeatedly. The results showed the chloroform: methanol (1:1) (LGd) fraction has most percentage of yield (59.96 %), anti-inflammatory activities, with IC50 of 23.94±0.71 µg/ml and antioxidant activities, with EC50 of 32.74±1.95 µg/ml.There were selected the most effective LG extract and fraction of anti-inflammatory and antioxidant activities to chemical constituents analysis by using gas chromatography-mass spectrometry (GC-MS). LG1 and LGd were isolated chemical compounds and identification to marker of that remedy. The results showed that Eugenol, Aceteugenol and Piperine showed the highest percentage of yield, as 31.22, 6.57 and 5.89 %, respectively. For LGd, the maximum percentage contents were (2E)-3-(2,3-Dimethoxyphenyl)-2-propenoic acid, 2,4-Di-tert-butylphenol and 3,4-Dimethoxyphenylbutadiene (DMPBD) as 34.62, 6.80 and 4.93 %, respectively.The stability testing of the ethanolic extract of LG (LG1) were investigated on accelerated condition as 40±2 OC with 75±5% RH for six months. After that, the LG1 was determined for quantity of anti-inflammatory activity on inhibitory effects on nitric oxide production. The results found that the ethanolic extract of Leard-ngam remedy show no significant difference of activity at the various times (p &gt; 0.05). Thus, it can be concluded that the ethanolic extract of Leard-ngam remedy was stable to inhibit NO production within 2 year. The active compound which isolated from LG remedy was Piperine. It was tested for inhibitory effect on Nitric oxide production, inhibition of PGE2 production. Piperine showed high inhibitory effect against Nitric oxide production and PGE2 production, with IC50 of 6.87±0.03, 5.78±0.54 µg/ml, respectively. These results were concluded that Piperine is an anti-inflammatory compound in the ethanolic extract of LG. These results support the use of Leard-ngam remedy to be anti-inflammation in dysmenorrhea and blood tonic because LG remedy and its herb components showed high inhibitory effect against NO production and strong radical scavenging activity