Online Submissions: http://www.journaltcm.com J Tradit Chin Med 2014 December 15; 34(6): 716-724 [email protected] ISSN 0255-2922 © 2014 JTCM. All rights reserved. EXPERIMENTAL STUDYTOPIC Effect of volatile oil from Blumea Balsamifera (L.) DC. leaves on wound healing in mice Yuxin Pang, Dan Wang, Xuan Hu, Hui Wang, Wanjin Fu, Zuowang Fan, Xiaolu Chen, Fulai Yu aa Yuxin Pang, Dan Wang, Xuan Hu, Xiaolu Chen, Fulai Yu, oil from Blumea Balsamifera (L.) DC. leaves (BB oil) Tropical Crops Genetic Resources Institute, Chinese Acade- on wound healing in mice. my of Tropical Agricultural Sciences, Danzhou 571737, Chi- na; Medicinal Efficacy Evaluation Laboratory, Hainan Provin- METHODS: Undiluted BB oil and its diluted solu- cial Engineering Research Center for Ainaxiang (Herba tions with olive oil to 1/5 and 1/10 to yield BB oil1/5 Blumeae Balsamiferae), Danzhou 571737, China; Key Labora- and BB oil were applied to the wounded skin be- tory of Tropical Crop Gene Resources and Germplasm En- 1/10 fore wound healing conditions were assessed by hancement of Ministry of Agriculture, Danzhou 571737, Chi- na. healing rate, histopathology, and contents of colla- Hui Wang, Wanjin Fu, College of Traditional Chinese Medi- gen, hydroxyproline, and Neuropeptide Substance cine, Guangdong Pharmaceutical University, Guangzhou P (SP). All above results were compared with the ef- 510000, China ficacies of the control, pure olive oil, basic fibro- Zuowang Fan, College of Traditional Chinese Medicine, blast growth factor (BFGF), and cream of Jing Wan Guangdong Pharmaceutical University, Guangzhou 510000, Hong (JWH). China; Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, RESULTS: BB oil and BB oil improved wound China; Key Laboratory of Tropical Crop Gene Resources and 1/5 1/10 contraction and closure. Histopathology study fur- Germplasm Enhancement of Ministry of Agriculture, Dan- zhou 571737, China ther confirmed a desirable histological organiza- Supported by the Fundamental Scientific Research Funds tion of wound tissues. BB oil1/5 and BB oil1/10 reduced for Chinese Academy of Tropical Agricultural Sciences — the number of inflammatory cells, increased Tropical Crops Genetic Resources Institute [The Evaluation wound-healing rates, and significantly increased and Mechanism Research for the Treatment of Ainaxiang the hydroxyproline content. Both BB oil and BB (Herba Blumeae Balsamiferae) Oil on Skin Burns Based on Cy- 1/5 oil improved formation of collagen, and reduced tokines, No. #1630032014016], the Natural Science Fund of 1/10 Hainan Province [the Evaluation and Mechanism Research the frequency of fibroblasts. Moreover, BB oil1/5 and of Ainaxiang (Herba Blumeae Balsamiferae) Oil on Healing BB oil1/10 markedly promoted SP expression. Howev- and Repairing of Skin Wounds, No. #312022], and the Natu- er, undiluted BB oil may induce skin thickening and ral Science Fund of China (Study on Variety Classification of hardening, inhibite collagen synthesis and delay "Nalong" and It's Ethanopharmacology of Li in Hainan, No. # complete skin wound healing. 81374065) Correspondence to: Dr. Yuxin Pang, Laboratory of Tropical CONCLUSION: The BB oil1/5 and BB oil1/10 promoted Medicinal Plants Resources, Tropical Crops Genetic Resourc- capillary regeneration, blood circulation, collagen es Institute, Chinese Academy of Tropical Agricultural Sci- ences, Danzhou 571731, China. [email protected] deposition, granular tissue formation, epithelial de- Telephone: +86-898-23300268 position, and wound contraction. The mechanism Accepted: September 5, 2014 underlying the action might be related to induc- tion of SP secretion, and the proliferation and differ- entiation of mesenchymal cells. Abstract OBJECTIVE: To assess the effectiveness of violate © 2014 JTCM. All rights reserved. JTCM | www. journaltcm. com 716 December 15, 2014 |Volume 34 | Issue 6 | Pang YX et al. / Experimental Study Key words: Wound healing; Neuropeptides; METERIALS AND METHODS Blumea Balsamifera (L.) DC.; Borneolum Synthciti- cum PLant and its volatile oil preparation Blumea Balsamifera (L.) DC. was obtained from Luo- dian, Guizhou, China. It was identified by Dr. Yuxin INTRODUCTION Pang, associate researcher fellow at the Tropical Crops Traditional medicine practitioners have described the Genetic Resources Institute, Chinese Academy of therapeutic efficacies of many traditional and indige- Tropical Agricultural Sciences, Danzhou, Hainan, Chi- nous plants against diseases.1 Natural products that are na. The herbarium voucher specimen (TCGRI 10011) safe, and possess physiological properties are excellent was saved. BB oil was obtained by distilling its sources of new therapeutics for the treatment of condi- air-dried leaves with steam at Guizhou Ai-Yuan Ecolog- tions like mechanical damage of the skin.2,3 Some re- ical Medicine Development Industry, Guizhou, China. searchers therefore have shifted their focus to the po- A yield of 0.1 mL of BB oil was obtained from each Kg tential wound healing properties of plants.4 of fresh Blumea Balsamifera (L.) DC. leaves. Wound healing is a process of restoring damaged cells and tissues.5-7 The phases of wound healing occur in a Determination of l-borneol content in BB oil precise and regulated order. Firstly, it involves hemosta- The standard l-borneol (98% , Batch No. 10147015) sis and inflammation; secondly, mesenchymal cells dif- was produced by Alfa Aesar, Heysham, Lancs, UK. ferentiate, proliferate, and migrate to the wound site; The content of l-borneol of BB oil was determined by thirdly, angiogenesis and epithelial deposition at the gas chromatography (GC, Agilent 7890, Santa Clara, wound surface site. Finally, synthesis, cross-linking and CA, USA), using ethyl acetate as the solvent. The sepa- alignment of collagen is required to provides structural ration was performed on a HP-5 quartz capillary col- strength to the wound healing tissue.8 The wound also umn (30 m×320 μm, 0.5 μm) equipped with a flame undergoes physical contraction, which might be medi- ionization detector (FID) detector. The injection vol- ated by contractile fibroblasts.9 Neuropeptide Sub- ume was 0.6 μL and was carried out by nitrogen with- stance P (SP) is a pro-inflammatory neuropeptide, and out splitting. The injector temperature was maintained modulates inflammatory responses of skin wounds. SP at 250 and detector, 280 . The oven temperature also promotes the synthesis and metabolism of fibro- was programmed℃ from 60 ℃for 1 min, raised to 100 blast and increases accumulation of collagen in the pro- at a rate of 5 /min, and℃ to 260 at a rate of 20 ℃/ liferative phase of mesenchymal cell growth and dy- min, and isotherm℃ at 260 for 5℃min. The number℃ of ℃ namics.10 In addition, SP is an important medium in theoretical plates was more than 50 000 and the separa- the process of wound repair and scar healing.11,12 tion degree was greater than 1.5. The external standard Ainaxiang (Herba Blumeae Balsamiferae), also named method was used. Sambong in some tropical countries, is an herb with a rich constitution of essential oils that are used in Tradi- Animals tional Chinese Medicine (TCM).13 In China, Blumea Ninety eight healthy Kun-Ming mice of specific patho- Balsamifera (L.) DC. normally grows in Hainan and gen-free (SPF) grade (No. SPF2012005) weighing (19± Guizhou.14,15 Its leaves have been used for healing many 1) g were supplied by the Experimental Animal Center conditions including eczema, dermatitis, skin injury, of Guangzhou University of Chinese Medicine [Certifi- skin bruises, beriberi, lumbago, menorrhagia, rheuma- cate of quality No. SCXK (Yue) 2008-0020], Guang- tism, and some other diseases.16 Recently, the extracts zhou, China. Forty nine female and forty nine male of the leaves have been verified to display physiological mice were maintained under 20 -25 with a relative activities on plasmin-inhibitory,17,18 anti-fungal,19,20 free humidity of 40%-70% and normal℃ photo℃ periods (12 h radical scavenging and anti-obesity functions.21,22 The light/12 h dark). The mice were individually housed Records of Seeking Herbal Medicines in Lingnan, an and fed a normal diet and allowed access to water ad li- ancient Chinese medical work, described that the ex- bitum in the Laboratory of Tropical Medicinal Plants tract from Ainaxiang (Herba Blumeae Balsamiferae), Resources, Tropical Crops Genetic Resources Institute, was used to treat snakebite injury, and skin wounds Chinese Academy of Tropical Agricultural Sciences, and itch. It documented that external application of Danzhou, China. This study was registered at and au- the mashed fresh leaves or leaf water washings decoc- thorized by Guangdong Pharmaceutical University, tion could treat traumatic injury, carbuncle and skin Guangzhou, China and Tropical Crops Genetic Re- pruritus.23 sources Institute, Chinese Academy of Tropical Agri- The main active ingredient in the violate oil from cultural Sciences, Danzhou, China (Trial registration: Blumea Balsamifera (L.) DC. leaves (BB oil) is l-borne- current controlled trials: GDPU 2012005). The experi- ol. However, little systematic study has been found on ment was conducted in accordance with the UK Ani- the pharmacologic use of BB oil. Herein, we have eval- mal Care and Control Act of 1986, and the National uated the potential for, and possible mechanisms of ac- Institutes of Health guide for the care and use of labo- tion of, BB oil to promote wound healing. ratory animals. JTCM | www. journaltcm. com 717 December 15, 2014 |Volume 34 | Issue 6 | Pang YX et al. / Experimental Study Animal modeling and grouping were cut out and immediately fixed in 10% v/v neu- Totally 98 mice were randomly divided into seven tral- formaldehyde for 24 h. The specimens were em- groups by random number table method: Control (n= bedded in paraffin and 4 μm sections were sliced for 14), solvent control (n=14), basic fibroblast growth fac- hematoxylin and eosin (HE) staining (HE staining kit, tor (BFGF) (n=14), cream of Jing Wan Hong (JWH) Nanjing Jiancheng Bioengineering Institute, Nanjing, (n=14), undiluted violate oil from Ainaxiang leaves China, batch No.