DOI: 10.14457/tu.the.2015.651
title: Anti-inflammatory and antioxidant activities of Thai traditional remedy for stroke called Lom-Am-Aa-Pruek and its plant ingredients

description: The Thai national list of essential medicine specifiesLom-Am-Ma-Pruek (LAMP) is Thai traditional medicine for stroke and an analgesic in the musculoskeletal system. It is in Chawadarn scripture, it consists of this medicinal plants as follows:  Allium sativum Linn, Alpinia galanga (L.) Willd, Cinnamomum camphora (L.) Presl, Cleome viscosa Linn, Crateva adansonii DC, Crate-va  religiosa G.Forst, Curcuma zedoaria (Berg) Roscoe, Erythrina variegata Linn, Myristica fragrans Houtt (seed and fruit), Piper nigrum Linn, Plumbago indica Linn, Zingiber cassumunar Roxb, Sodium Chloride (NaCl).When mixed together, the remedy has a spicy taste. Thai traditional medicine practitioners believed that spicy herbs can help balance the body. Therefore, LAMP should be investigated and confirmed as an anti-inflammatory preparation to support using it for treatment of stroke and muscle pain.Thus the, aims of this study were to investigate antioxidant activity by NBT assay and anti-inflammatory effects of LAMP and its plant ingredients. In this study, Griess’s reagent was used to measure the anti-inflammatory activity by inhibition effects of all extracts on nitric oxide production activated by lipopolysaccharide in RAW 264.7 cell lines. TNF-αand COX-2 inhibitor activities which cause inflammation in many organs were also determined foranti-inflammatory activity.  The chemical fingerprint of LAMP was analyzed by high performance liquid chromatography.LAMP remedy and its plant ingredients were extracted by two methods namely, maceration in 95% ethanol and decoction, to obtain ethanolic and aqueous extracts.  The yield of aqueous extractof LAMP was higher than the ethanolic extract (11.37% and 7.37%, respectively).  The ethanolic extract of Myristica fragrans Houtt (mace) showed highest percentage of yield (8.70%) andthe water extract ofAllium sativum Linn (42%) showed highest percentage of yield Raw material of plant ingredients were standardized by following Thai Herbal Pharmacopoeia standard (THP).  All plant ingredients of LAMP remedy met standard values except Cinnamomum camphora and Sodium Chloride.  However, LAMP remedy values all met THP criteria (the moisture content of LAMP remedy was 8.64±0.36%, total ash as 7.34±0.43% and acid insoluble ash as 1.76±0.05%).In vitro anti-inflammatory activities were tested by inhibition to NO production, inhibition effect on LPS-induced TNF- α release and LPS-stimulated PGE2 release on RAW 264.7 cell lines.  Antioxidant activity was by scavenging PMA-stimulated superoxide production in HL-60 cells measured by the NBT reduction.  For the LAMP remedy extracts, the 95% ethanolic extract (LAMPE) possessed potent inhibitory effect on NO production with IC50 value 24.90±0.86 μg/ml.  However, LAMPE exhibited less anti-inflammatory activity than Prednisolone (IC50 value 1.31±0.05 μg/ml). The aqueous extract of Lom-Am-Ma-Preuk remedy (LAMPW) exhibited weak anti-inflammatory activity on this path way (IC50 valuemore than 100 μg/ml). The ethanolic extract of Alpinia galanga showed the most effect on NO inhibitory assay (IC50 value 6.99±0.4 μg/ml).The second was the ethanolic extract ofCurcuma zedoaria (IC50 was value 14.38±1.4 μg/ml).  For the LAMP remedy extracts, the 95% ethanolic extract (LAMPE) possessed potent activity against TNF-α release but was less effective than prednisolone, a positive control with IC50 values 35.01±2.61 and 0.95±0.19 μg/ml, respectively. On the other hand, aqueous extract of LAMP remedy exhibited weak inhibitory TNF-αrelease activity.  The 95% ethanolic extract also showed the higher activity on PEG2 release thanthe aqueous extract (IC50 value 4.77±0.03 and more than 100 μg/ml, respectively). For ethanolic extract of plant ingredients, it was interesting that more than half of all extracts possessed potent inhibitory effect on LPS-stimulated PGE2 release. The ethanolic extract of A. galangal, M. fragrans (Mace), P. nigrum and Z. cassumunar exhibited the strongest potent on anti-inflammatory activity (IC50 values 1.23±0.01, 1.57±0.37, 2.95±0.49 and 3.08±0.34 μg/ml, respectively). It was not significantly different from positive control prednisolone IC50 value 0.96±0.01μg/ml (p-value&gt;0.05).  The second were most potent significantly different P. indica, M. fragrans (Nutmeg), E. variegate (IC50 values 4.09±0.32, 4.6±0.19 and 9.27±0.72μg/ml, respectively).  Theethanolic extract (LAMPE) had higher antioxidant activity by NBT assay than aqueous extract (LAMPW) with EC50 values 40.13±1.1 and more than 100 μg/ml, respectively.It was significantly different from positive control or propyl gallate EC50 value7.15±1.06μg/ml (p-value&lt; 0.05). A. galanga ethanolic extract exhibited the strongest antioxidant activity with EC50 value 3.94±0.35μg/ml.  It was not significantly different when compared with positive control or propyl gallate(p-value&gt; 0.05).Analysis by HPLC showed that myristicin as a compound in the ethanolic extract of LAMP showed the highest content (297.84±5.42 mg/g of extract). Next were piperine, eugenol and plumbagin (189.66±2.56, 58.75±0.13 and 45.01±2.35 mg/g of extract, respectively).  From this result, new knowledge and scientific report of chemical fingerprint and quantification of LAMP remedy and RP-HPLC method could be considered for quality control of LAMP extract.LAMP ethanolic extract was tested for stability under accelerated conditions (40±2oC with 75±5% RH for 6 months) and evaluated for anti-inflammation effect by inhibition of Nitric oxide production release from RAW 264.7 cell lines. The result revealed that anti-inflammation effect of the ethanolic extract  of LAMP by inhibition of nitric oxide production release from RAW 264.7 cell lines at Day15, 30, 60, 90, 120, 150 and 180 were not significantly different from day 0 (p-value&gt; 0.05).In conclusion, the 95% ethanolic extract of Lom-Am-Ma-Pruek remedy possessed potent anti-inflammatory activity on three pathways.  All of these findings support the traditional use of LAMP remedy for the treatment of stroke and an analgesic in the musculoskeletal system.  Thus, its ethanolic extract should be continuously studied in animal model for anti-inflammation, safety and product development using myristicinas an anti-inflamatory marker to analyse product.