DOI: 10.14457/tu.the.2018.1039
title: Anti-allergic and anti-inflammatory activities of Prasaprohyai and Benjakul remedies used for allergic rhinitis

description: Prasaprohyai and Benjakul remedies are Thai polyherbal formulation, which have been indicated in the list of herbal medicinal products of the national list of essential medicines of Thailand 2013. In Thai traditional medicine group, Prasaphohyai is used for treatments of fever, cold and allergy. It consists of nineteen plants namely rhizomes of Kaempferia galanga L., which the main component in 20 proportions and other components in equal proportions as each 1 proportion namely fruits of Amomum testaceum Ridl., fruits of Anethum graveolens L., roots of Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., roots of Angelica sinensis (Oliv.) Diels, all parts of Artemisia annua L., rhizomes of Atractylodes lancea (Thunb.) DC., fruits of Cuminum cyminum L., stems of Dracaena loureiroi Gagnep., fruits of Foeniculum vulgare Mill., seeds of Lepidium sativum L., rhizomes of Ligusticum sinense Oliv., flowers of Mammea siamensis (Miq.) T. Anders., flowers of Mesua ferrea L., flowers of Mimusops elengi L., stems, arils and seeds of Myristica fragrans Houtt., pollens of Nelumbo nucifera Gaertn., seeds of Nigella sativa L., and flowers of Syzygium aromaticum (L.) Merr. & L. M. Perry. Many of biological activities of Prasaprohyai have also been reported including anti-allergy, anti-inflammation, antibacterial, anticancer, etc. However, it has not been reported on in vivo anti-allergy. In part of Benjakul, it has long been used for balanced health, controlled abnormal of element in the body, relief of flatulence, carminative and adaptogenic drug before using other drugs. Benjakul consists of five plants in equal proportions namely stems of Piper interruptum Opiz., fruits of Piper longum L., roots of Piper sarmentosum Roxb., roots of Plumbago indica L., and rhizomes of Zingiber officinale Roscoe. Benjakul has been reported on adaptogenicity, antimalarial, cytotoxicity, etc. But it has not been reported on in vitro and in vivo anti-allergy and in vitro anti-inflammation. Thus, the objectives of present study aimed to evaluate Benjakul the in vitro anti-allergic activity using antigen-induced β-hexosaminidase release in RBL-2H3 cells, in vivo anti-allergic activity using OVA-induced allergic rhinitis reaction in BALB/c mice, and in vitro anti-inflammatory activity using lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor- (TNF-) in RAW 264.7 cells. For Prasaprohyai, it was focused only in vivo anti-allergic effect. The isolation of pure compounds from Atractylodes lancea which showed to be active plant ingredient was investigated by using vacuum liquid chromatography (VLC) and medium pressure liquid chromatography (MPLC) techniques. The stability of crude extract and capsule products from these remedies were also determined. Benjakul ethanolic extract exhibited the most potent β-hexosaminidase release with IC50 value of 12.69 g/ml and showed potent inhibitory activity on NO production with IC50 value of 16.60 g/ml, but not active on the release of TNF-. Moreover, Z. officinale and P. indica ethanolic extracts, plant components of Benjakul, represented the high inhibition on β-hexosaminidase release (IC50 = 12.93 and 13.31 g/ml, respectively) and also possessed the high potent effect on NO production (IC50 = 13.44 and 14.06 g/ml, respectively) and the inhibitory effect on NO production of these plants showed higher activity than Benjakul ethanolic extract. The ethanolic extract of P. interruptum exhibited the potential inhibition on TNF- release (IC50 = 21.84 g/ml). In addition, 6-shogaol and plumbagin, which are isolated compounds from Benjakul ethanolic extract, showed the strongest activities on both β-hexosaminidase release (IC50 = 0.28 and 4.03 g/ml, respectively) and NO production (IC50 = 0.92 and 0.002 g/ml, respectively) but only 6-shogaol exhibited the potent effect on TNF- release (IC50 = 9.01 g/ml). BALB/c mice were sensitized with ovalbumin (OVA) mixed with alum intraperitoneally and then treated with three doses of both Prasaprohyai and Benjakul and challenged intranasally with OVA. Prasaprohyai (100 mg/kg) treatment group reduced the number of grades of mucus production in nasal tissues. It also suppressed OVA-specific IgE and IgG1 serum levels and increased OVA-specific IgG2a level. Moreover, IL-4, IL-5 and IL-13 mRNA expressions in the nasal tissues of allergic mice were inhibited by treatment with Prasaprohyai ethanolic extract (100 mg/kg). The expressions of Th1 response (IL-12p35) and Treg response (IL-10) were slightly increased. However, the expressions of IFN-, IL-12p40 and TGF-β were unchanged in the nasal mucosa. As for Benjakul, serum OVA-specific IgE and IgG1 levels, the number of grades of inflammatory cells and mast cells of allergic mice lessened after treated with Benjakul ethanolic extract (37.5 mg/kg). In addition, Benjakul (37.5 mg/kg) treatment group also showed down-regulation of IL-5 and IL-13, which are Th2 cytokines and slightly up-regulated IL-10 mRNA expression. However, the expressions of IFN-, IL-12p35 and IL-12p40 were down-regulated and the TGF-β mRNA level was unchanged in nasal tissues.Three compounds including taraxerol acetate, isolated from Atractylodes lancea, atractylodin TS and β-eudesmol, authentic pure compounds from A. lancea, showed no effect on LPS-induced NO production in RAW 264.7 cells. Atractylodin TS showed strong inhibitory activity on antigen-activated β-hexosaminidase release in RBL-2H3 cells (IC50 = 0.07 g/ml) while β-eudesmol exhibited weak effect on this study (IC50 = 86.21 g/ml).The stability study after keeping under accelerated conditions (40ºC, 75% RH for 6 months) of Prasaprohyai ethanolic extracts was unaltered in β-hexosaminidase release and NO production inhibition. While, Benjakul ethanolic extract was unaltered in NO production but it altered β-hexosaminidase release at days 120, 150 and 180.Three formulations consist of one formulation of Prasaprohyai (P3) and two formulations of Benjakul (B1 and B4) were prepared in solid dosage form as capsule containing 10% w/w of extract, Avecel PH-101, Avecel PH-102, lactose, Aerosil, magnesium stearate and talcum having a total weight of 500 mg per capsule. Moreover, the weight variation and disintegration time of these capsules were performed in accordance with the BP 2013 standards. The stability test under accelerated conditions (40ºC, 75% RH for 6 months) of P3, B1 and B4 formulations were unchanged in the moisture content and NO production inhibition. Additionally, B1 and B4 formulations were unchanged in inhibiting β-hexosaminidase release but P3 formulation was changed in β-hexosaminidase release at days 150 and 180. The P3 formulation showed significantly increased on the release of β-hexosaminidase in RBL-2H3 cells when compared with day 0. These results can be concluded that Benjakul ethanolic extract showed the high effects on in vitro anti-allergic activity which inhibited the release of β-hexosaminidase and on in vitro anti-inflammatory activity which inhibited the NO production. Moreover, Benjakul and Prasaprohyai ethanolic extracts might be considered a potential therapeutic agent in treating allergic rhinitis. Benjakul ethanolic extract could be stored for one year at room temperature, due to the amount of plumbagin which rapidly changed. Therefore, B1 and B4 formulations could be stored for two years at room temperature without loss of activity. In addition, Prasaprohyai ethanolic extract could be stored for two years at room temperature while P3 formulation could be stored for 1.5 years at room temperature without loss of activity. Thus, these results may provide some scientific support for the use of Benjakul and Prasaprohyai for treatment of allergy-related diseases and inflammation-related diseases.