Blumea balsamifera—A Phytochemical and Pharmacological Review

The main components of sambong (Blumea balsamifera) are listed in this article. The whole plant and its crude extracts, as well as its isolated constituents, display numerous biological activities, such as antitumor, hepatoprotective, superoxide radical scavenging, antioxidant, antimicrobial and anti-inflammation, anti-plasmodial, anti-tyrosinase, platelet aggregation, enhancing percutaneous penetration, wound healing, anti-obesity, along with disease and insect resistant activities. Although many experimental and biological studies have been carried out, some traditional uses such as rheumatism healing still need to be verified by scientific pharmacological studies, and further studies including phytochemical standardization and bioactivity authentication would be beneficial.


General Botanical Description
According to the description of Flora Republicae Popularis Sinicae and Chinese Materia Medica [19,20], B. balsamifera is a perennial herb or subshrub, which rises about 1-3 meters in height. Its stem is strong and taupe, and erects with taupe, longitudinal edges. Its upper internodes are covered by dense tawny nonglandular hair. Its leaves, when triturated, send out a unique, cool aroma, which can make people feel refreshed. The leaves are wide ovoid or oblong-lanceolate in shape at the bottom, 22-25 cm in length, 8-10 cm in width. Its base is attenuated with petiole, narrow linear appendants of 3-5 pairs on both sides, pubescenced above, slight brown or thick yellow-white silky-villous, highlighted below midrib, with lateral veins of 10-15 pairs. The leaves at the top are oblong-lanceolate or ovate-lanceolate in shape, 7-12 cm in length, 1.5-3.5 cm in width, with an acuminate apex, a slightly acuminate base, without petiole or with a short petiole with narrow linear appendants of 1-3 pairs, entire or with thin serration or pinnatopectinate. Capitulum is arranged in much more branched leafy panicles; a peduncle with yellow and dense pubescence; an involucre campanulate, and a dense pubescence at the back. Its flower is yellow, with numerous female parts; tubular corolla, receptacle honeycomb, and corolla thin tubulous. Akene is cylindrical, with five edges, a dense pubescence, and a red-brown rough hairy pappus. The flowering period almost covers the whole year. B. balsamifera often grows in forest edges, under forests, river beds, valleys, or grasslands, and the altitude is 600-1000 m. In addition to its various Chinese locations it is also distributed in India, Pakistan, Burma, Indo-China Peninsula, Malaysia, Indonesia and Philippines.

Phytochemistry
There have been more than 100 volatile or non-volatile constituents isolated from sambong, including monoterpenes, sesquiterpenes, diterpenes, flavonoids, organic acids, esters, alcohols, dihydroflavone, and sterols. The study of the plant mainly focused on the volatile oils and flavonoids, which possessed various bioactivities in vivo and in vitro. The chemical constituents of B. balsamifera have been reviewed earlier [8,18].

Volatile Constituents
The volatile constituents account for the largest amount of the constituents in B. balsamifera, which are the major active constituents containing terpenoids, fatty acids, phenols, alcohols, aldehydes, ethers, ketones, pyridines, furans, and alkanes (Table 1, Appendix A). The most important constituent is L-borneol. To address the need for a natural borneol, different extraction methods have been used to extract the volatile constituents from B. balsamifera. Steam distillation (SD), simultaneous distillation and extraction (SDE), and CO 2 supercritical extraction were the most common methods in the extraction of volatile oils [21][22][23][24]. Wang et al. have adopted SD, SDE, and headspace solid-phase micro-extraction (HS-SPME) in order to obtain the volatile compounds of B. balsamifera leaves [23]. The extracts were then isolated and were identified by gas chromatography mass spectrometry (GC-MS). They found that alcohols and terpenoids were the main volatile compounds and the terpenoids accounted for a considerable proportion. Fifty, twenty-four, and forty-nine kinds of compounds were extracted by SD, SDE, and HS-SPME, respectively.  Several volatile oils are contained in the leaves and branchlets of B. balsamifera, which are the key crude materials of refined borneol [28]. Volatile oil in B. balsamifera is a yellow oily liquid with a unique aroma [32]. The oil yield of this plant is at least 2.5 mg/g (DW) in Guizhou, China. If the plants are fertilized in certain way, it can be much higher, may be up to 7.72 mg/g (DW) [33]. In Hainan (China), Pang et al. also got an oil yield of 3.2-4.3 mg/g (unpublished data). Some previous studies have reported that the volatile oils of B. balsamifera mainly contained monoterpenes and sesquiterpenes, such as L-borneol, 10-epi-γ-eudesmol, γ-eudesmol, β-eudesmol, α-eudesmol, limonene, L-camphor, palmitic acid, and D-camphor [21]. Hao et al. have qualitatively and quantitatively analyzed the volatile oil of B. balsamifera growing in Guizhou Province by GC-MS [25]. A total of 28 constituents were identified in this study, including: L-borneol, β-caryophyllene, camphor, γ-eudesmol, 1-octen-3-ol, trans-β-ocimene, and 1,3,4,5,6,7-hexahydro-2,5,5-trimethyl-2H-2,4a-ethanonaphthalene.

Sterols
Apart from the above constituents, a small number of sterols were also isolated from B. balsamifera. Zhao et al. obtained colorless acicular as well as sheet crystals from B. balsamifera by silica gel column chromatography [56], where the crystals were identified to be stigmasterol and β-sitosterol by TLC and melting point measurement. Chen isolated β-sitosterol, daucosterol, and 5α,8α-epidioxyergosta-6,22-dien-3β-ol from the aerial sections of B. balsamifera collected from Mengla, Yunnan by MS determination [9]. Liang et al. yielded seven compounds, which were isolated from ethyl acetate and chloroform extract, including daucosterol [39].

Sesquiterpene Lactone (SLs)
Sesquiterpene lactones (SLs) are a group of common chemicals in many Asteraceae plants. They were famous because they had cytotoxic and potential to be tumor inhibitors [57,58]. In sambong, a member of Asteraceae family, there were three sesquiterpene lactones, Blumealactone A, Blumealactone B, and Blumealactone C. Fujimoto et al. isolated them by extracting its dried leaves with 90% ethanol [54].

Other Constituents
There were some other constituents in this plant. Chen found two coumarin constituents, such as umberlliferone and hydranngetin, in B. balsamifera. He also found a lignans constituent, which was syringaresinol [9].

Antitumor Activity
Hasegawa et al. extracted a dihydroflavonol from B. balsamifera as a result of screening among more than 150 plant materials [12]. The dihydroflavonol components showed the most significant synergism with tumor related apoptosis inducing ligand (TRAIL). It enhanced the level of TRAIL-R2 promoter activity and promoted the expression of surface protein in a p53-independent manner. The ethanol extract of B. balsamifera leaves was tested on male mice to investigate its hepatoxicity. The results exhibited that the hepatic cells, sitplasm, nucleus, and sinusoid of the mice liver were damaged through some changes in the liver color and texture [59]. The methanol extract of B. balsamifera inhibited the growth in rat and showed no cytotoxicity on human hepatocellular carcinoma cells. The methanol extract decreased the expression of cyclin-E and phosphorylation of retinoblastoma (Rb) protein resulting in cell cycle arrest. Likewise, it decreased the level of the proliferation related ligand (APRIL) [60,61]. Moreover, the methanol extract of B. balsamifera was used to determine its cytotoxicity on a panel of human cancer cell lines by MTT assay. There was no regular or acute cytotoxicity on the cells of HepG2, HCT-116, T-47D, NCl-H23 and CCD-18Co [62]. Saewan et al. found six compounds out of nine isolated flavonoids to have cytotoxicity against KB, MCF-7, and NCI-H187 cancer cell lines [14]. These six compounds were evaluated for cytotoxicity against KB, MCF-7, and NCI-H187 cancer cell lines. Three compounds were active against the KB cells with the IC 50 values of 17.09, 47.72, and 17.83 µg/mL, respectively. Another three compounds exhibited a moderate activity against the NCI-H187 cells with the IC 50 values of 16.29, 29.97, and 20.59 µg/mL. Luteolin-7-methyl ether showed a strong cytotoxicity against human lung cancer (NCI-H187) cell lines with an IC 50 of 1.29 µg/mL and a moderate toxicity against oral cavity cancer (KB) cell lines with an IC 50 of 17.83 µg/mL. Li et al. studied the antitumor activity determined by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay [13]. The three endophytic streptomycetes strains of B. balsamifera, including: YIM 56092, YIM 56093, and YIM 56099 exhibited anticancer activity. Yet, different strains displayed different antitumor activities. The YIM 56092 strain displayed a cytotoxic activity on polyketide synthases I (PKS-I) nonribosomal peptide synthetases (NRPS) and P388D1. The YIM 56093 strain displayed a cytotoxic activity on PKS-Ⅱ, NRPS, and P388D1. The YIM 56099 was on the PKS-I, PKS-II, and NRPS. Fuijimoto et al., extracted blumealactone A, B, and C from sambong's dried leaves and found them could inhibit the growth of Yoshida sarcoma at the concentration of 5-10 μg/ml [54]. Lee disclosed a medication combination including sambong (Ainaxiang) and found it could enhance the efficiency of curing hepatoma and pancreatic cancer treatments [63].

Hepatoprotective Effects
Xu et al. demonstrated that oral blumeatin (5,3',5-trihydroxy-7-methoxydihydroflavone) exhibited a significant protective activity against the liver injury caused by paracetamol and prednisolone [64]. Furthermore, Xu and Zhao have shown that five other blumea flavanones possessed protective activity for acute experimental liver injury [65]. Pu et al. further verified the five blumea flavanones protecting the hepatocytes against lipid peroxidation, which was induced by CCl 4 or FeSO 4 +cysteine. Certain concentration of the five compounds (10-100 μmol/L) inhibited the malonaldehyde production, GSH depletion, and GPT leakage of hepatocytes [66]. Furthermore, blumea flavanone II showed the strongest activity. They also reported that the blumea flavones had protective effects against acute liver injury induced by different chemicals [67].

Anti-Microbial and Anti-Inflammation Activity
Ongsakul et al. claimed that the crude aqueous and ethanolic extracts of B. balsamifera displayed no significant antibacterial activity against the strains of Staphylococcus aureus and Escherichia coli [73]. However, the stain of B. balsamifera, including YIM 56092 and YIM 56093, displayed a significant activity against S. epidermidis, such that YIM 56099 was active against E. coli. There seems to be no antimicrobial activity against S. aureus, Klebsiella pneumonia, and Candida albicans [13]. Chen isolated twelve new compounds [9], four of which displayed inhibitory activities against LPS-induced NO production in RAW 264.7 with the IC 50 [74]. Furthermore, the hexane extract inhibited Enterobacter cloacae and S. aureus. These results suggested that the extracts of B. balsamifera possessed an activity against certain kinds of infectious and toxin-producing microorganisms. It could potentially be utilized to prevent and treat microbial diseases.

Antiplasmodial Activities
According to the traditional efficacy of relieving fever, the methanol extract of B. balsamifera from Forest Research Institute Malaysia was investigated for any potential antiplasmodial activity. The extracts of roots and stems exhibited some activity against Plasmodium falciparum D10 strain (sensitive strain) with an IC 50 value of (26.25 ± 2.47) μg/mL and (7.75 ± 0.35) μg/mL, respectively [75].

Antityrosinase Activities
The ethylacetate extract consisting of nine flavonoids were isolated from the leaves of B. balsamifera. Their antityrosinase activities were surveyed by Saewan et al. [14]. According to their reports, compared with arbutin, two dihydroflavonols, dihydroquercetin-4'-methylether and dihydroquercetin-7,4'-dimethylether, and three flavonols, quercetin, rhamnetin and tamarixetin, showed a significantly higher inhibitory activity, but another two flavanones, 5,7,3',5'-tetrahydroxyflavanone and blumeatin, and two flavones, luteolin and luteolin-7-methyl ether, showed a lower inhibitory activity. The possible mechanism of the antityrosinase activity might be the cause of chelating with copper in the active center of tyrosinase.

Platelet Aggregation Activities
The concentration of 1.26 μmol/L blumeatin displayed a significant promoting activity on the rat and human platelet aggregation caused by arachidonic acid, 5-hydotypamice, and epinephrine. However, concentrations of 0.315 and 2.52 μmol/L inhibited platelet aggregation. It suggested that the effects of blumeatin on the platelet aggregation were dependent upon the concentration used. The injection of B. balsamifera extracts decreased the blood pressure, expanded the blood vessels, and inhibited the sympathetic nervous system in order to address the high pressure and insomnia. The infusion of the plant also had the function of diuresis [67].

Enhancing Percutaneous Penetration Activity
The L-borneol, as the main effective compound of B. balsamifera, showed a percutaneous penetration enhancer effect. The essential oil camphor and 1-menthol of the plant specifically promoted the percutaneous absorption of nicotinamide [9]. Fu et al. further verified the 0.5%, 1.0%, and 2.0% B. balsamifera oil enhancing albuterol sulfate transdermal absorption, respectively [76]. The percutaneous penetration of a combination of 1.0% B. balsamifera oil and 1.0% azone was less than that of their separate uses.

Wound Healing Activity
Wang et al. discovered that the external application of B. balsamifera oil on the intact and damaged skin of rats exhibited no acute toxicity [77]. The rats with pure B. balsamifera oil exposure at dosage of 2000 mg/kg for 24 h showed no allergic reaction or acute toxicity reaction, but a better wound recovery activity as compared to the one treated with non-B. balsamifera oil formulations. The results were consistent with the traditional use in ethnic minority, Li and Miao, in China in order to heal the skin wound and itch [19].

Anti-Obesity Activity
Kubota et al. reported that the extracts of B. balsamifera inhibited the lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activities [17], which mainly decreased the expressions of key adipogenic transcription factors, such as peroxisome proligerator-activated receptor γ, CCAAT element binding protein, and leptin in the 3T3-L1 adipocytes. Therefore, the extracts of B. balsamifera might possess antidiabetic, antiatherogenic, and anti-inflammatory functions. The methanol extracts of B. balsamifera (100 μg/mL) were used to investigate the ability of inhibiting blood vessel's formation by the method of rat aortic ring. The results exhibited that there was no remarkable differences between B. balsamifera extracts and vehicle control treatment [62].

Disease and Insect Resistant Activity
Luo et al. reported that the acetone extracts of B. balsamifera possessed an activity against Pryicutaria oryzae, Fusarium oxysporum sp., Colletorichum musae, C. gloeosporioides, C. capsici, and F. oxysporum f. sp. in vitro, with an inhibition rate of over 90.0% [78]. The volatile oil of B. balsamifera inhibited Aeromonas hydrophila, F. graminearum, and Magnaporthe grisea [27]. Wang et al. also demonstrated that the extract of B. balsamifera leaves showed a 60.8% insecticidal activity against the adult Aleurodicus disperses [23]. Furthermore, the essential oil of B. balsamifera showed fumigant toxicity against the maize weevils, such as Sitophilus zeamais [26]. The crude oil also induced the death in the S. zeamais adults. The results suggested that the extracts of B. balsamifera possessed significant disease and insect resistant activities, and could be used as new potential plant pesticides.

Conclusions
In traditional medicine, the source of the plant should be primarily clear and definite. Thus, the general botanical description should be reviewed according to the Flora Republicae Popularis Sinicae and Chinese Materia Medica [19,20]. The herbal authentication of B. balsamifera was then further carried out to verify the plant source and medicinal efficacy [19]. These studies confirmed the accuracy and uniqueness of the source of B. balsamifera. In the same way, as a folk medicine, B. balsamifera has been widely used in South Asian countries, especially in China. Due to the chemical constituents contained in this plant for the different effects, the phytochemical research has been reviewed in this article. The chemical constituents were mostly volatile and non-volatile. The former constituents occupied the largest amount, which mainly included: terpenoids, fatty acids, phenols, alcohols, aldehydes, ethers, ketones, pyridines, furans, and alkanes. L-borneol was the most abundant and active constituent in this plant, while other constituents included flavonoids, sterols, sesquiterpene lactones, and other constituents [8,18]. The chemical composition of B. balsamifera oils varies from different plant populations [22,25,26,28], indicating more studies should be done on individual's structural development, the cultivation, geographical, and climate conditions and essential oil standardization. The survey and summary of the extensive studies revealed that B. balsamifera was an essential and valuable medicinal plant used for folk treatments such as treating eczema, dermatitis, beriberi, lumbago, menorrhagia, rheumatism, skin injury, or used as insecticide [45]. As a traditional medicine, the biological and pharmacological studies of the plant materials, crude extracts, and isolated chemical constituents of B. balsamifera offered experimental and scientific proofs for its various traditional uses. The pharmacological studies focused on studying the anti-microbial and anti-inflammatory effects [13,15], antiplasmodial effects [75], platelet aggregation [79], wound healing [77], and disease and insect resistant activities [26], all of which confirmed the plant's traditional uses. Moreover, some new pharmacological uses were discovered, such as antitumor [12][13][14], hepatoprotective [66], superoxide radical scavenging [16], antioxidant [69], antityrosinase [14], enhancing percutaneous penetration [76], and anti-obesity activities [17]. However, there was no experimental and pharmacological evidence to prove the traditional uses of this plant in rheumatism and lumbago. Besides, the isolated chemical constituent and its correspondent pharmacological effects were rarely simultaneously carried out in one study. Hasegawa et al. extracted a dihydroflavonol from B. balsamifera, which exhibited a significant synergism with TRAIL by pharmacological experiment [12]. Nevertheless, more studies should be done in abundance to understand the pharmacodynamic chemical constituents. The outcome from this study could establish the basis for its future clinical utilization in modern science.
On the basis of the above review, several prospects were revealed. In order to further define the effective chemical compounds, the biological activities of monomeric compounds, the plant material, and its crude extraction further studies were proposed. In addition, some traditional effects of B. balsamifera, such as rheumatism still need to be testified by more modern methods and further pharmacological trials. Few more aspects such as pharmacokinetics, molecular biology, and naturel medicinal chemistry should be utilized to study its phytochemical standardization and bioactivity identification according to its bioactive metabolism. Moreover, in the production process of TCM Aipian, B. balsamifera oil and Aifen could be important accessory substances yielded, which also possess many chemical and biological activities to be further studied in the future. Therefore, we concluded that the area of B. balsamifera research should be significantly expanded. 57

Conflicts of Interest
The authors declare no conflict of interest.