Original Article ENHANCEMENT OF STABILITY, RELEASE AND IN VITRO DIGESTIBILITY OF MULBERRY STEM EXTRACT USING MICROEMULSIONS NATTAPORN SOONTHORNSIT 1 , WARINKARN HEMSTAPAT 2 , CHETSADAPORN PITAKSUTHEEPONG 3 , TASANA PITAKSUTEEPONG 1* 1 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Tha Pho, Mueang Phitsanulok, Phitsanulok 65000, Thailand, 2 Department of Pharmacology, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand, 3 Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand Email: tasanap@nu.ac.th Received: 21 Mar 2017 Revised and Accepted: 19 May 2017 ABSTRACT Objective: This study was aimed to develop oral microemulsions and to evaluate their ability to enhance stability, release and intestinal digestion of mulberry stem extract (MSE). Methods: The pseudoternary phase diagrams were constructed using caprylic/capric triglyceride (oil), PEG-8 caprylic/capric glycerides (S), polyglyceryl-3 diisostearate (CoS) and an aqueous phase. The effects of S/CoS (Km) ratio and a cosolvent, i.e. polyethene glycol 400 or propylene glycol (PG), were investigated. The optimized formulations were selected and incorporated with MSE. Then, they were then subjected to stability, release and lipolysis studies. The control solution consisted of 50% PG and 50% water. Results: The formation and characteristics of the microemulsions were influenced by Km and cosolvents. The two optimized formulations (F3 and F4) consisted of 10% oil, 70% S/CoS mixture and 20% aqueous phase were chosen. The Km ratios of F3 and F4 were 4:1 and 3:1. The aqueous phase of F3 and F4 was water and water mixed with PG, respectively. These formulations could improve the stability of MSE better than the control solution. The accumulated release of MSE from F3, F4 and the control solution reached 100% while that of unformulated crude extract reached only 70% after 6 h. The lipolysis study showed that MSE incorporated in both F3 and F4 was digested more than double the percentage compared to that of MSE incorporated in the control solution. Conclusion: MSE was successfully developed in microemulsions. They are shown to be promising vehicles for oral delivery of MSE. Further animal trials are suggested. Keywords: Mulberry stem extract, Microemulsion, Stability, Release, In vitro intestinal digestion © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2017v9i7.18609 INTRODUCTION Various parts of the mulberry tree have long been widely used in Ayurvedic and various traditional medical systems [1]. The root bark is used as anti-tussive, anti-inflammatory and diuretic agents in Korea [2]. The leaves, fruit and bark have been used in traditional Chinese medicine to treat fever, improve eyesight, strengthen joints and lower blood pressure [3, 4]. The leaves are also consumed in Korea, Japan and Chile as anti-hyperglycemic nutraceutical foods for patients with diabetes mellitus [5]. Recently, the ethanolic extract prepared from aerial parts of the mulberry tree has been shown to provide hypolipidemic, anti-inflammatory and antioxidative effects which helped in alleviating obesity-induced liver diseases and related metabolic disorder in rats [6]. However, the mulberry stems which are often pruned and cut as normal cultivation practice are rarely investigated for medicinal use. Our previous study demonstrated the anti-inflammatory effects of MSE through the inhibition of nitric oxide, inducible nitric oxide synthase (iNOS) and cyclooxygenase (Cox)-2 productions in LPS- activated RAW 264.7 macrophages [7]. In addition, this extract has been investigated for antioxidant activities using various in vitro antioxidant assays [8]. It was shown to possess a hydrogen-donating ability, hydroxyl radicals quenching ability, superoxide scavenging activity, nitric oxide scavenging activity and iron reducing capacity [8]. It also has been investigated for the anti-nociceptive effect in anterior cruciate ligament transection (ACLT)-induced rat model of osteoarthritis (OA) [9]. Assessment of knee joint pain was performed by determining the change in the hind limb weight distribution between the operated and the non-operated contralateral limb using the hind limb weight bearing tester. The severity of cartilage damage to the knee joint on the operated limb was also observed using the modified Mankin grading system. It was found that oral administration of MSE at doses of 5.6, 56 and 560 mg/kg for 8 w significantly alleviated joint pain in a dose-dependent manner. MSE at 560 mg/kg also demonstrated to delay the progression of the articular cartilage degeneration. Considering these background studies, MSE has potential to be used as a dietary supplement for reducing OA pain or an alternative treatment for inflammatory and oxidative stress-related diseases. The benefit of using plant crude extracts is that they contain complex mixtures of compounds which often contribute to the synergistic effect [10]. The stem of the mulberry tree has recently been reported to contain various polyphenolic constituents including flavonoids (morusin), stilbenoids (mulberroside, resveratrol and oxyresveratrol) and coumarins [2]. However, these polyphenols are also sensitive to heat and a lack of long-term stability [11, 12]. From our previous study, the effect of temperature on the stability of pure compound oxyresveratrol and MSE was investigated [12]. It was observed that the degradation occurred as a function of temperature and time. The degradation of oxyresveratrol and the extract was more pronounced at high temperature (45 °C) and was greatly observed after 45 d of storage. In addition, the solubility of mixtures of compounds is also a major challenge. The polyphenolic compounds are known to have poor oral bioavailability due to low aqueous solubility and low dissolution rate [13]. According to the biopharmaceutics classification system (BCS), most of polyphenols belong to class II (low solubility and high permeability) and class IV (low solubility and low permeability) [13]. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 9, Issue 7, 2017