Comparative Phytochemical Analysis and Antibacterial Efficacy of in Vitro and in Vivo Extracts from East Indian Sandalwood Tree

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/231610036

Comparative Phytochemical Analysis and Antibacterial Efficacy Of In Vitro And In Vivo Extracts From East Indian Sandalwood Tree.

ARTICLE in LETTERS IN APPLIED MICROBIOLOGY · SEPTEMBER 2012 Impact Factor: 1.66 · DOI: 10.1111/lam.12005 · Source: PubMed

CITATIONS READS 11 205

2 AUTHORS:

Biswapriya Biswavas Misra Satyahari Dey University of Florida IIT Kharagpur

52 PUBLICATIONS 110 CITATIONS 69 PUBLICATIONS 667 CITATIONS

SEE PROFILE SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Biswapriya Biswavas Misra letting you access and read them immediately. Retrieved on: 21 March 2016 Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE Comparative phytochemical analysis and antibacterial efﬁcacy of in vitro and in vivo extracts from East Indian sandalwood tree (Santalum album L.)

B.B. Misra1,2 and S. Dey1

1 Plant Biotechnology Laboratory, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, Midnapore (West), West Bengal India 2 Present address: Center for Chemical Biology, Universiti Sains Malaysia [CCB@USM], 1st Floor Block B, No 10, Persiaran Bukit Jambul, 11900 Bayan Lepas, Pulau Pinang, Malaysia

Significance and Impact of Study: This study constitutes the first systematic investigation on phytochemical composition and antimicrobial efficacy of sandalwood tree across in vitro and in vivo developmental stages screened against thirteen bacterial strains by four methods. Using a battery of antimicrobial assay techniques, it is possible to follow the differential bioactive metabolic richness of plant parts, to decipher, for example comparable efficacy of somatic embryo extracts and sandalwood oil.

Keywords Abstract antibacterial, extracts, in vitro, phytochemical, sandalwood oil, santalol, Santalum album, Sandalwood oil has been found in numerous therapeutic applications in TLC bioautography. traditional medicines such as Chinese traditional medicine and Ayurveda. However, there are no comparative accounts available in the literature that Correspondence focused on in vitro and in vivo tree sample-derived extracts. Combined B.B. Misra, Plant Biotechnology Laboratory, dichloromethane and methanol extracts were obtained from in vitro samples, Department of Biotechnology, Indian Institute that is, callus, somatic embryo and seedlings, and in vivo from leaves of non-oil- of Technology Kharagpur, Midnapore (West) Kharagpur-721302, West Bengal, India. yielding young and oil-yielding matured trees. Phytochemical evaluation of the E-mail: [email protected] extracts reveals that the tree is rich in terpenoids, saponin, phenolics and tannins. The antibacterial properties of the five extracts were compared with sandalwood 2012/1333: received 25 July 2012, revised 6 oil by screening against nine Gram-negative and five Gram-positive bacterial September 2012 and accepted 23 September strains by disc diffusion, agar spot and TLC bioautography methods. Minimum 2012 inhibitory concentration (MIC) for sandalwood oil was determined to be in the range of 0078–5 lgml 1 for most of the test micro-organisms screened. doi:10.1111/lam.12005 Bioautography results indicated the presence of potential antimicrobial constituents in somatic embryo extracts and sandalwood oil. Among the extracts screened, the somatic embryo extracts showed the strongest antibacterial activity comparable only with sandalwood oil and matured tree leaves’ extract. The findings presented here also suggest that apart from sandalwood oil, other parts of this tree across developmental stages are also enriched with antibacterial principles.

matured tree ranges from 25 to 6%, which is the highest Introduction among all known 20 Santalum species, depending on the The East Indian sandalwood tree, Santalum album L., is age of the tree and colour of the heartwood (Shankaran- identiﬁed as one of the most important medicinal tree in arayana and Kamala 1989). The estimated global annual the world (Fox 2000). This woody tree is a root hemipara- requirement is about 10 000 tons of wood, equivalent to site tropical species belonging to the taxonomic group 200 tons of oil, involving a trade of about $360 million, of Santalaceae and yields precious sandalwood, which con- which only 10% is met from natural resources. The quality tains over 90% of santalols, a mixture of sesquiterpenoids and composition of essential oil obtained from young and and hence is the focus of many investigations (Demole matured sandal trees varies among heartwoods and sam- et al. 1976). The essential oil yield from a 50-year-old pling heights (Shankaranarayana and Parthasarathi 1987).

Sandalwood oil is used in Indian traditional medicine extracts (8–10%, w/w) and was the lowest in callus at 4%. system Ayurveda, as antiseptic, antipyretic, antiscabietic, The ash content of the five tissues showed variability diuretic, expectorant, stimulant and for the treatment of depending upon their in vitro and in vivo origin. bronchitis, dysuria, urinary infection, gonorrhoeal recov- ery owing to its antibacterial and antifungal properties Phytochemical evaluation of sandalwood extracts (Handa et al. 1951; Okasaki and Oshima 1953; Winter 1958; Dastur 1962; Jain 1968; Dikshit and Hussain Phytochemical analyses suggest that the extracts were rich 1984). The hydrolysed exhausted sandalwood powder on in terpenoids (monoterpenoids and sesquiterpenoids), pharmacological screening demonstrated antiremorogen- saponins, phenylpropanoids such as phenolics, proantho- ic, anti-inflammatory, antimitotic, antiviral, anticancer- cyanidins, flavonoids, phenolics, condensed tannins, o- ous, anti-hypertensive, antipyretic and sedative properties quinines and polyphenols (Table S1). (Desai et al. 1991). The oil also possesses antiviral activ- Sesquiterpenoid standard curve (y = 0002x, ity against certain herpes simplex virus types (Benencia R2 = 0988) was obtained for quantification purpose and Courreges 1999). Recently, the major constituent of using bulk-purified a-santalol as a standard. Total sesquit- oil, a-santalol, was proven to be effective as a skin can- erpenoid content in in vitro plant extracts (173– cer chemopreventive agent (Kaur et al. 2005). Moreover, 514mgg 1 extract) was found to be higher than that of it was shown that various organic fractions and sesquit- in vivo extracts (8–374mgg 1 extract). Total monoterp- erpenoid compounds from the oil possess anti-Helicob- enoid content was quantified using an l-linalool acter pylori properties, which is the causative organism (y = 00001x, R2 = 0932) standard curve and was found for gastric cancer and peptic ulcer (Takaishi et al. 2005). to be similar for the extracts (3–45mgg 1 extract) and Epidemic phytoplasmal ‘spike’ disease leading to severe the highest for young tree leaves’ extract (95mgg 1 destruction, illegal poaching and over exploitation owing extract). Total saponin content (diosgenin standard curve, to the increased global demand are the reasons of it being y = 0017x 0197, R2 = 0988) of callus, somatic inducted into IUCN, Red List of Threatened Species as embryo and seedling extracts was comparable (94– vulnerable (IUCN 2006). Unsurprisingly, the first in vitro 171mgg 1 extract), whereas the in vivo extracts showed micropropagation study on any woody forest plant was higher saponin contents (316–436mgg 1 extract). reported for sandalwood, that is, callusing from embryos Phenolics are the most plentiful classes of constituents (Rangaswamy and Rao 1963) followed by somatic in the plant kingdom (Rao 2003). In sandalwood phy- embryogenesis, regeneration, suspension cultures, somatic toextracts, 18–190 mg gallic acid equivalents (GAE) g 1 embryo production and maturation in air lift bioreactors of total phenolics was recorded. The highest phenolics (Das et al. 1998). Furthermore, in vitro callus is known content recorded for dietary constituents is spinach to yield sandalwood oil constituents (Yamashita 1997). (c.071 mg g 1), followed by swamp cabbage With the developmental stages of the plant very well (c. 041 mg g 1), and cabbage (c. 011 mg g 1; Ismail established and its age old medicinal properties being et al. 2004). Tannins are widely distributed among the confirmed recently by advanced means, this study was angiosperms. Furthermore, tannins such as gallic acid, undertaken to probe the antibacterial properties of non- catechins and tannin-glycosides were reported in the cal- polar extracts from in vitro samples, that is, callus, lus cultures of Quercus acutissima (Tanaka et al. 1995). somatic embryo and seedlings, and in vivo from the The fact that saponins are represented in sandalwood leaves of non-oil-yielding and oil-yielding matured tree extracts (94–436mgg 1 extract) is further corroborated leaves with sandalwood oil. by the fact that the plant order Santalales are known to To our knowledge, this is the first time effort towards contain oleane-type triterpenoid saponins (Crespin et al. the evaluation of biological activities of the nonpolar 1993). In grape marc extracts, anthocyanins are present in extracts from this particular tree, as a comparative study 068 mg g 1 extract (Vatai et al. 2008), comparable with across both in vitro and in vivo stages. the anthocyanin content in sandalwood (001–031 mg g 1 extract). Calli of common hawthorn, Crataegus monogyna, produce proanthocyanidins (1074–2959 mg g 1 DW), Results and discussion while the fruits and flowers produce proanthocyanidins (009–2028 mg eq. cyanidins g 1 DW) (Froehlicher et al. Physicochemical properties of sandalwood tissues and 2009). Sandalwood cells and tissues showed the presence extracts of good quantities of flavonoids. Several flavonoids have The moisture content of in vitro tissues (93–95%) is signifi- also been identified from Santalum insulare leaves, that is, cantly higher (P < 001) than the in vivo-grown trees (59– chlorogenic acid, luteolin, apigenein and apigenin- and 74%), while the extract yield was similar for the four luteolin-glucopyranosides (Butaud et al. 2006).

Table 1 Correlations between groups of metabolites quantified in Table 2 MIC values (lgml1) obtained for sandalwood oil against the five extracts. Groups 1 and 2 represent the groups of metabolites 14 bacterial strains (both Gram +ve and Gram ve) determined by quantified and plotted against each other to obtain the correlation the microtitre well plate-based broth twofold dilution method

Equation, Serial Bacterial strains and

Serial correlation no. NCIM codes MIC50* MIC70* MIC90* no. Group 1 Group 2 coefficients (R2) Gram-negative 1 Total phenolics Total casein-bound y = 2059x, 1 Acinetobacter 5 †† tannins R2 = 0999 calcoaceticus, 2 Total phenolics Total BSA-bound y = 1937x, 2886 tannins R2 = 0961 2 Alcaligenes 5–10 >40 † 3 Total phenolics Total PVPP-bound y = 0825x, faecalis, tannins R2 = 0989 2105 4 Total phenolics Total saponins y = 4669x, 3 Citrobacter 125 5 † R2 = 0834 fruendii, 5 Total phenolics Total anthocyanins y = 0183x, 2488 R2 = 0970 4 Escherichia 0156 0312 5–10 6 Total saponins Total BSA-bound y = 2281x, coli, 2931 tannins R2 = 0778 5 Klebsiella 0312–0625 0625–125 10 7 Total saponins Total PVPP-bound y = 3761x, aerogenes, tannins R2 = 0787 2098 8 Total saponins Total casein-bound y = 2259x, 6 Pseudomonas 25 †† tannins R2 = 0829 aeruginosa, 9 Total monoterpenoids Total flavan-3-ols y = 001x, 2945 R2 = 1 7 Pseudomonas 125 5 † fluorescens, 2100 ††† Indexed values of metabolites revealed that with the 8 Pseudomonas developmental progression, the metabolic diversity of putida, each metabolic group increased quantitatively. The 2174 9 Salmonella 0078 10 † amount of uncharacterized metabolite pool represented in typhimurium, the extracts is as follows: old tree leaves (33 33%) birches revealed that levels of phenolics, hydro- 12 Enterobacter 10 †† lysable and condensed tannins and flavonoids shared cloacae, strong positive correlations (Riipi et al. 2004). Interest- 2164 ingly, the saponin contents were correlated with the levels 13 Micrococcus ††† of tannins (BSA, PVPP and casein bound) and phenolics. flavus, 2376 Moreover, antifungal constituents such as terpenoid and 14 Staphylococcus 0078 0156–0312 0625 phenolic glycosides are known to be induced and accu- aureus, 5021 mulated in cereals challenged with arbuscular mycorrhizal fungus, Glomus intraradices (Maier et al. 1995). Similarly, *Values are the average of three measurements. it was observed that the flavan-3-ols, monoterpenoids and †Values not obtained. flavonoids contents are strongly correlated (Table 1). 1 >40 lgml . The lowest MIC50 values were observed against Salmonella typhimurium (0078 lgml 1) and the Antimicrobial efficacy of sandalwood extracts 1 highest MIC50 against Enterobacter cloacae (10 lgml ). Microtitre well plate-based broth dilution assay results The lowest MIC90 value was observed against Staphylococ- 1 (Table 2) revealed that the Gram-negative bacterium cus aureus (00625 lgml ) and the highest MIC90 was Pseudomonas fluorescens and the Gram-positive bacterium against Klebsiella aerogenes (10 lgml 1). This experiment Micrococcus flavus were resistant to sandalwood oil. The provided a measure of experimental concentrations to be highest MIC70 was recorded against Alcaligenes faecalis at used for the sandalwood oil and the extracts for the

Table 3 Antibacterial activity of tissue extracts based on disc diffusion method. Inhibition zones are given in mm units. Kanamycin (positive control, 30 lg disc)

Microbial Sl. Somatic Young Sandalwood susceptibility no. Bacterial strain Callus embryo Seedling tree Old tree oil Kanamycin index (%)

Gram-negative 1 Acinetobacter ** **528 ± 04522 ± 08285 ± 1820 calcoaceticus, 2886 2 Alcaligenes ** **523 ± 0665 ± 09245 ± 2120 faecalis, 2105 3 Citrobacter ** *** 65 ± 12182 ± 320 fruendii, 2488 4 Escherichia coli, 2931 ** *** 62 ± 05228 ± 220 5 Klebsiella ** **** 236 ± 260 aerogenes, 2098 6 Pseudomonas ** ** 95 ± 1567 ± 04188 ± 0820 aeruginosa, 2945 7 Pseudomonas 123 ± 24146 ± 2673 ± 0992 ± 1895 ± 21103 ± 22242 ± 12 100 ﬂuorescens, 2100 8 Pseudomonas * 91 ± 21 ** 64 ± 3894 ± 13175 ± 2640 putida, 2174 9 Salmonella * 78 ± 21 *** 122 ± 3166 ± 3220 typhimurium, 2501 Gram-positive 10 Arthrobacter ** *** 189 ± 15236 ± 460 nicotianae, 2460 11 Bacillus subtilis, 2063 * 523 ± 09 *** 72 ± 23195 ± 2320 12 Enterobacter 15 ± 2513± 299 ± 2284 ± 17115 ± 1394 ± 21224 ± 37 100 cloacae, 2164 13 Micrococcus * 72 ± 03104 ± 2 * 54 ± 14111 ± 11226 ± 2360 ﬂavus, 2376 14 Staphylococcus 171 ± 23218 ± 44 *** 185 ± 23246 ± 3840 aureus, 5021 Activity (%) 25 5625 3125 25 5625 9375 *

*No activity detected/obtained. determination of antibacterial activity against the same that is Arthrobacter nicotianae and Kl. aerogenes, did not show pool of micro-organisms. During the course of experi- any sensitivity to any of the extracts. ments, a well manifested ‘paradoxical effect’ was also From the agar spot assay results, it was revealed that observed, whereby a higher dose of sample was found to (Table 4), the lowest MSI was demonstrated by Acineto- be less bacteriostatic/inhibitory than a lower dose (Lacey bacter calcoaceticus and Ps. fluorescens (both, 0%), both and Lord 1981). Gram-negative strains, thus rendering them the most The disc diffusion assay results (Table 3) revealed the high- resistant strains, whereas the highest MSI was observed est microbial susceptibility index (MSI) against Ent. cloacae against Pseudomonas aeruginosa (100%). Among the (100%) and Ps. fluorescens (100%). The lowest MSI was Gram-positive bacterium, the highest MSI was observed obtained for Arthrobacter nicotianae and Kl. aerogenes (both, for A. nicotianae (80%) and the lowest for M. flavus 0%). The highest activity was recorded for sandalwood oil (0%). The highest activity was observed for sandalwood (9375%) and the lowest for both callus and young tree leaves’ oil (9285%) and the lowest for old tree leaves’ extract extracts at 25%. Somatic embryo and old tree leaves’ extracts (1428%), thus making them the most and the least bio- showed comparable activities. Kl. aerogenes displayed the low- active extracts used in this investigation. Moreover, the est MSI overall at 0% MSI and with susceptibility only against callus and the somatic embryo extracts showed the high- kanamycin. The highest sensitivity to sandalwood oil was est sensitivity towards the pathogenic Gram-positive bac- observed against A. nicotinae, while somatic embryo extracts terium Staph. aureus, at comparable zones of inhibition, displayed highest activity against Staph. aureus. Two bacteria, that is c. 125 mm. Seedling extract displayed the highest

Table 4 Agar spot detection method for antibacterial activity of the tissue extracts and sandalwood oil

Microbial Sl. Somatic Young Old Sandalwood susceptibility no. Extracts Callus embryo Seedling tree tree oil Kanamycin index (%)

Bacterial strains and NCIM codes: Gram-negative 1 Acinetobacter ** ***105 ± 14284 ± 230 calcoaceticus, 2886 2 Alcaligenes 70 ± 1484 ± 1273 ± 0772 ± 09* * 262 ± 1580 faecalis, 2105 3 Citrobacter 81 ± 118± 0973 ± 0774 ± 12* 108 ± 32185 ± 3880 fruendii, 2488 4 Escherichia 91 ± 21106 ± 1482 ± 1173 ± 11* 82 ± 21228 ± 2780 coli, 2931 5 Klebsiella *75 ± 12* 76 ± 13* 97 ± 14243 ± 2740 aerogenes, 2098 6 Pseudomonas 8 ± 02118 ± 0975 ± 058± 1273 ± 0594 ± 14173 ± 34 100 aeruginosa, 2945 7 Pseudomonas ** ***146 ± 172528 ± 5250 ﬂuorescens, 2100 8 Pseudomonas *75 ± 09* * 63 ± 1117 ± 14225 ± 2240 putida, 2174 9 Salmonella *78 ± 21* * * 122 ± 3166 ± 3220 typhimurium, 2501 Gram-positive 10 Arthrobacter 62 ± 0569 ± 0967 ± 1161 ± 12* 60 ± 05239 ± 4680 nicotianae, 2460 11 Bacillus subtilis, 74 ± 0975 ± 256± 1* * 10± 1019± 3360 2063 12 Enterobacter 86 ± 2182 ± 1262 ± 13* * 105 ± 33238 ± 2160 cloacae, 2164 13 Micrococcus ** ***11± 1231 ± 20 ﬂavus, 2376 14 Staphylococcus 126 ± 21128 ± 33* * * 526 ± 015 19 ± 2540 aureus, 5021 Activity (%) 5714 7857 50 4285 1428 9285

*No activity detected/obtained. activity against Escherichia coli (82 ± 11 mm). Young mum antimicrobial efficacy against most microbes was and old tree leaves’ extracts demonstrated maximum demonstrated by the compound with Rf value of 02– activity against Ps. aeruginosa, whereas sandalwood oil 025, previously identified as a-santalol (data not shown). was the most active against Gram-negative Ps. fluorescens Most instances of inhibition was found against C. freundii (146 ± 17 mm) and the most ineffective against Gram- (24 no’s) for various extracts. Sandalwood oil displayed 2 positive A. nicotinae (62 ± 05 mm). maximum area of inhibition against E. coli (52mm, Rf, The TLC-bioautography-based antibacterial activity 02), callus and somatic embryo extracts against Citrobact- 2 2 screening (Table 5) indicated the maximum number of er fruendii [(185 mm , Rf,053) and (334 mm , Rf, constituents to be active against M. flavus (5), followed 053)], seedling extract against Pseudomonas putida 2 by Ps. aeruginosa and Citrobacter freundii (4 each). Maxi- (124 mm , Rf,023), and young and old tree leaves’

Table 5 TLC bioautography antibacterial assay of ﬁve extracts and sandalwood oil, against the 13 bacterial strains. Rf denotes the position of the compounds on the HPTLC plate, and the area denotes the zone of inhibition/clear zone as found out for the speciﬁc band

Sandalwood Somatic oil Callus embryo Seedling Young tree Old tree

Area Area Area Area Area Area 2 2 2 2 2 2 Serial no Bacterial strain, NCIM code, gram character Rf (cm ) Rf (cm ) Rf (cm ) Rf (cm ) Rf (cm ) Rf (cm )

Gram negative 1 Acinetobacter calcoaceticus, 2886 008 062 002 028 002 035 051 095 0 02008 045 023 189**** * #008 058 022 098 2 Alcaligenes faecalis, 2105 023 28002 045 002 063**** 0178 063 134**** ** 063 088 063 121 3 Arthrobacter nicotianae, 2460 023 415 * * 004 066 002 023 * * 023 314 038 013**** ** ** ** 077 253**** ** ** 077 01 4 Citrobacter fruendii, 2488 004 048 04098 04135 * * 008 122 0 021 02187 053 185 053 334 * * * 022 152 03022**** ** ** ** 063 031**** ** ** 063 021 5 Escherichia coli, 2931 0252****023 124 021 311 008 354 6 Klebsiella aerogenes, 2098 024 34* * 007 026**** ** 0881********081 102 7 Pseudomonas aeruginosa, 2495 005 03002 027 003 036 * * 008 012 * * 023 35********023 283 053 03********** 063 05******063 081 063 08 8 Pseudomonas ﬂuorescens, 2100 021 279**** ** ** ** 053 084**** ** 053 11* * 9 Pseudomonas putida, 2174 023 262 * * 005 094 043 125 * * 023 189 053 016**** ** ** 053 012 Gram positive 10 Bacillus subtilis, 2063 02346 014 045 014 056 * * 008 112 015 289 05054 * * 056 108**** ** 058 095**** ** ** 053 065 078 069**** ** ** ** 11 Enterobacter cloacae, 2164 0226008 152 008 254 * * 0 122 * * 12 Micrococcus ﬂavus, 2376 0 21********** 023 312**** 033 109 * * 023 152 038 019**** ** 053 034 * * 053 038**** ** ** 088 279 063 03********** 13 Staphylococcus aureus, 5021 023 274**0095**** **

*No activity detected/obtained. extract against E. coli, at comparable areas of inhibition lar constituents from the extracts. Substantial investiga- 2 2 [(311 mm , Rf,021) and (354 mm , Rf,008)]. Interest- tions have proven the antimicrobial properties of ingly, the assay results with Gram-positive strains were Australian sandalwood oil (Beylier and Givaudan 1979). not too encouraging as compared with their Gram-nega- In this particular study, sandalwood oil yielded MIC val- tive counter parts that showed good sensitivities to the ues mostly oscillating in the range of 03–10 lgml1, bioactive constituents. thus indicating the sesquiterpenoid-rich sandalwood oil as The optimized broth dilution method using 002% a strong bacteriostatic agent, as suggested by the classiﬁ- Tween 80 to emulsify the oils, by serial broth dilution cation of plants based on MIC values (i.e. up to method, has been developed and shown to be the most 500 lgml 1 as ‘strong’; Aligiannis et al. 2001). Interest- accurate method for testing the antimicrobial activity of ingly, the ethanolic extracts of sandalwood seeds were the hydrophobic and viscous essential oils (Hood et al. shown to have no antibacterial properties (Patil et al. 2003). In this study, similar guidelines were followed to 2011), thereby indicating that the seeds do not accumu- address the issues of solubility of essential oil and nonpo- late such principles, whereas the aqueous extracts from

Letters in Applied Microbiology 55, 476--486 © 2012 The Society for Applied Microbiology 481 Antibacterial principles of sandalwood B.B. Misra and S. Dey leaves and stem did show antimicrobial potential (Kumar duced by specialized cells and/or at distinct developmen- et al. 2006). It is well known that a combined nonpolar tal stages (Balandrin et al. 1985). Besides, the extract of dichloromethane and methanol is enriched with development of a certain level of differentiation is also phenolics and the terpenoid fractions of the plant mate- considered to be important in the successful production rial, which was also further revealed by HPTLC (high per- of phytochemicals by cell cultures. Furthermore, it is well formance thin layer chromatography) analyses (data not established that woody plant responses to stress and shown). Phenolics and terpenoids are proven antibacterial environmental cues are driven by plant demands for compounds. In the previous studies, heartwood-extracted particular types of compounds rather than by changes in essential oil was shown to have maximum inhibitory resource availability (Koricheva et al. 1998). The results activity against Bacillus mycoides and E. coli (Chourasia also demonstrated that some compounds are probably 1978). not synthesized if the cells remain undifferentiated, that is Sandalwood oil constituents and their synthetic ana- callus, as observed elsewhere (Berlin et al. 1985). logues are known to be strongly antimicrobial (Viollon et Thus, the observed definitive development-associated Chaumont 1994) and antibacterial (Jirovetz et al. 2006). increase in antibacterial properties may be attributed to Moreover, Staph. aureus is one of the most susceptible maturation and differentiation processes and, hence, with bacterium to plant extracts (Chariandy et al. 1999), as an assumption that similar constituents may be contribut- observed in this case, against sandalwood extracts. San- ing to antibacterial properties. The controlled and undif- dalwood essential oil is active against all of the bacterial ferentiated states in vitro may not have induced the plant strains except Pseudomonas aeruginosa, which is also system to produce sufficient antimicrobials, but as the resistant to other essential oils (Skaltsa et al. 2003). In plant is exposed to more stressful events of differentiation the TLC bioautographic assay, the antibacterial activity of and such other environmental cues, the synthesis and sandalwood oil constituents, that is a-santalol and b-san- secretion of antibacterial constituents becomes a regular talol, is demonstrated. In fact, santalols are known to be phenomenon. Nevertheless, the gradual fall in antibacterial active against Salm. typhimurium and Staph. aureus, spe- values of the extracts beyond the somatic embryo stage, cifically epi-b-santalene (Simanjuntak 2003). Apart from that is the in vitro seedling and the field-grown plant stage, the sesquiterpenoids, the proanthocyanidins (Heinonen remained unexplainable. Thus, it was demonstrated that 2007) and phenolic constituents (Rodriguez Vaquero biological activity screening can provide simpler tools to et al. 2009) are known to be responsible for the antimi- investigate the physiological status and developmental crobial properties of phytoextracts. In fact, essential oils stage and address several questions like enrichment of are also known for their strong in vitro and in vivo anti- metabolic diversity concurrent with development. fungal properties that are associated with the diverse Distinctly, the somatic embryo stood out of the rest of array of terpenoid constituents (Mondello et al. 2006). the extracts against the Gram-negative strains, whereas Highly nonpolar constituents like santalbic acid (trans- sandalwood oil is highly bacteriostatic as evident from 11-octa-decen-9-ynoic acid) from sandalwood seeds have activity against most of the strains used in the study. The shown to be effective against some Gram-positive bacteria more interesting aspect to be studied would be the syner- and pathogenic fungi (Jones et al. 1995). Generally speak- gistic effect of such extracts and to identify the molecules ing, the small organic hydrophobic phytochemicals that are specifically responsible for the responsible anti- extracted with the solvents confer antimicrobial proper- bacterial activity. Further investigations underway at the ties to the sandalwood extracts. Besides, it is well estab- author’s laboratory would provide insights about the rela- lished that cytoplasm membrane coagulation, breakdown tion of the plant secondary metabolism with the associ- of protons motive force, electron flux and active trans- ated biological activities. port unbalance are critical events responsible for provid- ing the antimicrobial property to phytochemicals (Sikkema et al. 1995; Carlson et al. 2002). Materials and methods A close correlation exists between the expression of secondary metabolism and morphological and cytological Plant materials differentiation. However, it is not yet clear to what extent In vitro-grown callus, somatic embryo and the seedlings secondary metabolism depends on the development of were cultured and maintained by subculturing at intervals of specific structures, and it remains to be established if 3–6 weeks, in the laboratory from a highly proliferating cell these two processes are genetically and/or physiologically line (IITKGP/91) on solid media, that is, Woody Plant linked (Wiermann 1981). The biggest challenge of pro- Media (WPM; Lloyd and McCown 1981) + 2,4-D ducing secondary metabolites from plant cell suspension (1 mg l 1); WPM + IAA (05mgl 1) + BAP (05mgl 1); cultures is that secondary metabolites are usually pro-

and WPM + IBA (05mgl 1) + BA (05mgl 1) + GA Quantitative phytochemical investigation (05mgl 1), respectively, supplemented with 3–4% sucrose and 025–05% Phytagel (Sigma, St Louis, MO, USA). A 7- Crude phytoextracts were subjected to qualitative and year-old non-oil-yielding tree and a 15-year-old oil-yielding quantitative phytochemical analyses to detect the major matured tree, growing in the Department of Biotechnology, chemical groups present (Harborne 1984; Kokate et al. Indian Institute of Technology, Kharagpur campus, were 1998; Heinonen 2007), and the values were expressed as sampled for fresh leaves. mg g 1 extract.

Bacterial strains Determination of MIC The bacterial strains of NCIM type collections were The MIC (in lgml 1) values of sandalwood oil were deter- obtained from the National Chemical Laboratory (Pune, mined by serial broth dilution method using the high- India), for example Gram-negative strains: Klebsiella aer- throughput sterile 96-well microtitre round-bottom/flat- ogenes (2098), Pseudomonas aeruginosa (2945), Pseudomo- bottom plate screening method (Hood et al. 2003). Sandal- nas fluorescens (2100), Acinetobacter calcoaceticus (2886), wood oil was dissolved to a stock concentration of Escherichia coli (2931), Citrobacter fruendii (2488), Bacillus 8 lgml 1 (i.e. specific gravity equivalent to 1 at 25°C) in subtilis (2063), Alcaligenes faecalis (2105) and Salmonella 01% Tween 80. Aliquots of 100 ll were added to the mic- typhimurium (2501), and Gram-positive strains: Enterobacter rotitre plate wells containing 100 ll of Mueller-Hinton cloacae (2164), Arthrobacter nicotianae (2460), Staphylococcus (MH) broth, and continuing with the twofold serial dilu- aureus (5021) and Micrococcus flavus (2376), and main- tion series, spanning the ten columns, such that the con- tained on nutrient broth (NB) media and regularly centrations of sandalwood oil were 00078–4% (v/v) subcultured from their glycerol stocks stored in 80°C. corresponding to 0078–40 lgml 1 as the final concentration. Aliquots (5 ll) of freshly grown over night bacterial inoculums were added to all except the first blank well and Extraction of nonpolar fractions a negative control with only 002% Tween 80 in MH media. Briefly, around 100 g of each of the five types of material Plates were incubated at 37 ± 1°C under shaking condi- was collected and dried overnight in an oven at 60°C and tions 180 rev min 1, for 16 h in an incubator (New Bruns- ground finely into powder in a mortar and pestle using wick Scientific Co., New Brunswick, NJ, USA). After liquid nitrogen, and it was extracted for 18 h under reflux incubation, the plates were read with an ELISA reader in a heating mantle, with a mixture of dichloromethane/ (Bio-Rad, Hercules, CA) at 595 nm as well as by visual methanol (1 : 1) (v/v) (Balick 1994) at 40°C, in a ratio of detection, and data were recorded, by deducting the optical 1 : 200 (w/v) of plant material and solvent (20 l). Postex- density (OD) values generated against blank and negative traction, the solid particulate matter was excluded by control wells. The MIC50, MIC70 and MIC90 values were filtration in vacuum using Whatman No.1 filter papers, fol- defined as the lowest dilution of essential oil required that lowed by centrifugation at 3000 g for 15 min. The obtained inhibited visible growth by 50, 70 and 90%, respectively, clear organic fractions were further concentrated in with respect to the control growth. The tests were carried vacuum using a rotary evaporator (Eyela N–N series; Rika- out in three duplicate plates to yield reproducibility. kikai Inc., Tokyo, Japan) at 60°C. The individual extracts (8–10 g) obtained were stored in 80°C, while for con- Disc diffusion method for antibacterial screening ducting the antimicrobial assays, the extracts were dissolved in 01% Tween 80 (200 mg ml 1) and stored at 4°C for Antibacterial activity was screened against 13 bacterial short-term use. Authenticated sandalwood oil was obtained strains by disc diffusion method (Bauer et al. 1966). The from Cauvery (Bangalore, India) and was tested by HPTLC bacterial strains stored as glycerol stocks in 80°C deep (Camag, Muttenz, Switzerland) for compositional analyses. freezer were subcultured in 5 ml of MH media, at 37°C, 180 rpm overnight in a shaker (New Brunswick Scientific Co.). The following day, cultures were streaked on 15% Physicochemical characterization of tissues and extracts agar supplemented MH plates and the single colonies were The obtained extracts were analysed with a pH meter freshly grown over night as mentioned earlier. A volume of (720 A; Orion, Bosta, MA). The yield of evaporated dried 2 9 105–106 colony forming units (CFUs), that is 100 llof extracts based on dry weight basis was calculated from the bacterial cell suspension, was taken for uniform spreading following equation: Yield (%) = (W1 9 100)/W2, where on MH agar plates (9 cm). Sterile paper discs of 5 mm W1 was the weight of extract after evaporation of solvents diameter were punched from Whatman No. 3 filter papers and W2 was the dry weight of the fresh plant sample. and soaked in appropriate amounts of organic extracts/

sandalwood oil dissolved in 002% Tween 80 (E. Merck, backed TLC plates 60 F254 (10 9 10 cm) of 250 lm thick- Darmstadt, Germany). The discs were applied on the lawn ness (E. Merck). Samples redissolved in dichloromethane/ of bacteria and were allowed to grow for 16 h at 37 ± 1°C, methanol (1 : 1) (100 lg for five extracts and 1 lg for san- and the clear inhibition zones were measured by a zone- dalwood oil) were loaded using a 100-ll sample-loading measuring scale (HiMedia, Mumbai, India) for antibacte- syringe (Hamilton, Bonaduz, Switzerland), at an applica- rial potential. Kanamycin discs (30 lg) (HiMedia) were tion rate of 01 lls 1, up to ten samples per plate, 4 mm used as positive control, while 01% Tween 80-loaded discs apart, with a band length of 8 mm per track/lane. Linear served as negative controls. The 5-mm discs as such or with ascending development was done in a 10 9 10 cm twin DMSO (dimethyl sulphoxide) were also applied onto the trough chamber, mobile phase: cyclohexane : ethyl acetate plates as blank or negative control. The assays were per- (9 : 1 ratio), in 4 ml volume. The development chamber formed in triplicates to minimize error. was supersaturated, and the chromatoplate development was carried out in the dark. Optimized chamber saturation time was 20 min, and the approximate linear ascending Agar spot method for antibacterial activity development time was about 12 min at 25 ± 3°C. The Antibacterial activities were measured by agar spot chromatographic plates were run up to 9 cm, until maxi- method (NCCLS 2000). Bacterial lawn was prepared as mum number of bands got resolved. described earlier, but instead of discs, test samples 2 ll Freshly prepared spray reagent [ethanolic solution of (c. 10 lg) and antibiotic, that is kanamycin, 2 ll p-anisaldehyde (25%), sulphuric acid (35%) and glacial (c. 10 lg) were spotted onto the lawn of bacteria. acetic acid (16%)] specific for sesquiterpenoids was used for derivatization and detection of phytoconstituents for plates run in parallel for phytochemical characterization. Quantitative evaluation of antibacterial activity A Scanner 3 (Hamilton, Bonaduz, Switzerland), with slit Antimicrobial efficiency as comparative numerical values dimension at 6 9 045 mm and a scanning speed of are generally recorded in terms of MIC (lgml 1), total 10 mm s 1, was used for densitometric scanning. Densito- activity values activity (%) values and MSI (Rangasamy metric analysis of plate was performed at the absorbance– et al. 2007). reflectance mode of 208 nm (determined absorption maxima for santalols), apart from the full UV range scan- Activityð%Þ¼100 ½No. of susceptible strains to a ning from 190 to 350 nm (D2-deuterium lamp). Analysis software was winCATS software (ver. 1.3.4.80.913.15; specific extract/Total no. of tested Camag). Compounds were identified on the basis of their microbialstrains Rf values of authenticated standards, maximum absorption The per cent activity (%) demonstrates the total anti- spectrum and literature. Evaluation was via peak areas with microbial potency of particular extracts. It shows that the linear regression. Only compounds with 95% levels of pur- number of microbial strains found susceptible to one par- ity were considered for quantification and identification. ticular extract.

Acknowledgements Microbial susceptible index (MSI) ¼ 100 ½No. of extracts effective against each microbial strain/ The authors would like to thank Mr. Rahul Nahar for his assistance in a few experiments. B.B.M. received the No. of total samples screened Junior and Senior Research Fellowships from the Council MSI is used to compare the relative susceptibility among of Scientific & Industrial Research (CSIR), New Delhi, the microbial strains. MSI values ranges from ‘0’ (resistant and Research Associateship conferred by the Department to all samples) to ‘100’ (susceptible to all samples). of Biotechnology (DBT), Government of India. The experimental work in Santalum album in the author’s laboratory is being supported under the project ‘Prospecting TLC bioautography for antibacterial activity of novel genes and molecules of S. album L.’, sponsored by DBT, Government of India. Thin layer chromatography bioautography for the screening of antibacterial compounds was performed as described previously (Nostro et al. 2000). A Camag (Hamilton, Conflict of Interest Switzerland) workstation and the sample applicator, Linomat 5 (Camag), were used for the HPTLC run. The The authors have declared that there is no conflict of stationary phase used was precoated silica gel aluminium- interest.

References Demole, E., Demole, C. and Enggist, P. (1976) A chemical investigation of the volatile constituents of East Indian Aligiannis, N., Kalpotzakis, E., Mitaku, S. and Chinou, I.B. sandalwood oil (Santalum album). Helv Chim Acta 59, (2001) Composition and antimicrobial activity of the 737–747. essential oils of two Origanum species. J Agric Food Chem Desai, V.B., Hiremath, R.D., Rasal, V.P., Gaikwad, D.N. – 40, 4168 4170. and Shankarnarayana, K.H. (1991) Pharmacological Balandrin, M.F., Klocke, J.A., Wurtele, E.S. and Bollinger, W. screening of HESP and sandal oils. Indian Perfumer H. (1985) Natural plant chemicals: sources of industrial 35,69–70. – and medicinal materials. Science 228, 1154 1160. Dikshit, A. and Hussain, A. (1984) Antifungal action of some Balick, M.J. (1994) Ethnobotanical screenings of medicinal essential oils against animal pathogen. Fitoterapia 55, 171–176. plants most often yield higher hit rates than random Fox, J.E. (2000) Sandalwood: the royal tree. Biologist 47,31–34. screenings. Ethnobotany, drug development and Froehlicher, T., Hennebelle, T., Martin-Nizard, F., biodiversity conservation exploring the linkages. Ciba Cleenewerck, P., Hilbert, J., Trotin, F. and Grec, S. (2009) Found Symp 185,4–18. Phenolic profiles and antioxidative effects of hawthorn cell Bauer, A.W., Kirby, W.M., Sherris, J.C. and Turk, M. (1966) suspensions, fresh fruits, and medicinal dried parts. Food Antibiotic susceptibility by a standardized single disk Chem 115, 897–903. method. Am J Clin Pathol 45, 493–496. Handa, K.L., Kapoor, L.D., Chopra, I.C. and Somnath, S. Benencia, F. and Courreges, M.C. (1999) Antiviral activity of (1951) Present position of crude drugs used in indigenous sandalwood oil against herpes simplex viruses-1 and -2. medicine. Indian J Pharm 13, 28. Phytomedicine 6, 119–123. Harborne, J.B. (1984) Phytochemical Methods: A Guide to Berlin, J., Beier, H., Fecker, L., Forche, E., Noe´, W., Sasse, Modern Techniques of Plant Analysis. London: Chapman F., Schiel, O. and Wray, V. (1985) Conventional and and Hall. new approaches to increase the alkaloid production of Heinonen, M. (2007) Antioxidant activity and antimicrobial plant cell cultures. In Primary and secondary effect of berry phenolics-a Finnish perspective. Mol Nutr metabolism of plant cell cultures ed. Neumann, K.H., Food Res 51, 684–691. Barz, W. and Reinhard, E. pp. 272–280. Berlin: Hood, J.R., Wilkinson, J.M. and Cavanagh, H.M.A. (2003) Springer-Verlag. Evaluation of common antibacterial screening methods Beylier, M.F. and Givaudan, S.A. (1979) Bacteriostatic activity of utilized in essential oil research. JEssentOilRes15, 428–433. some Australian essential oils. Perfum Flavor 4,23–25. Ismail, A., Marjan, Z.M. and Foong, C.W. (2004) Total Butaud, J.F., Raharivelomanana, P., Bianchini, J.P., Faure, R. and antioxidant activity and phenolic content in selected Gaydou, E.M. (2006) Leaf C-glycosylflavones from Santalum vegetables. Food Chem 87, 581–586. insulare (Santalaceae). Biochem Syst Ecol 34,433–435. IUCN. (2006) Asian Regional Workshop 2006. (Conservation Carlson, C.F., Mee, B.J. and Riley, T.V. (2002) Mechanisms of and Sustainable Management of Trees, Vietnam) 1998, action of Melaleuca alternifolia (tea tree) oil on Santalum album L. In: IUCN Red List of Threatened Staphylococcus aureus determined by time kill, lysis, leakage Species. (www.iucnredlist.org) and salt tolerance assays and electron microscopy. Jain, S.K. (1968) Medicinal Plants. pp. 123–125. New Delhi: Antimicrob Agents Chemother 46, 1914–1920. National Book Trust. Chariandy, C.M., Seaforth, C.E., Phelps, R.H., Pollard, G.V. Jirovetz, L., Buchbauer, G., Denkova, Z., Stoyanova, A., Murgov, and Khambay, B.P.S. (1999) Screening of medicinal I. and Gearon, V. (2006) Comparative study on the plants from Trinidad and Tobago for antimicrobial antimicrobial activities of different sandalwood essential oils and insecticidal properties. J Ethnopharmacol 64, 265– of various origin. Flavour Fragr J 21, 465–468. 270. Jones, G.P., Rao, K.S., Tucker, D.J., Richardson, B., Barnes, A. Chourasia, O.P. (1978) Antibacterial activity of the essential and Rivett, D.E. (1995) Antimicrobial activity of santalbic oils of Santalum album and Glossogyne pinnatifida. Indian acid from the oil of Santalum acuminatum (Quandong). Perfume 22, 205–206. Int J Pharm 33, 120–123. Crespin, F., Olliver, E., Lavaud, C., Babadjamian, A., Faure, R., Kaur, M., Agarwal, C., Singh, R.P., Guan, X., Dwivedi, C. Debrauwer, L., Balansard, G. and Boudo, G. (1993) and Agarwal, R. (2005) Skin cancer chemopreventive Triterpenoid saponins from Opilia celtidifolia. agent, {alpha}-santalol, induces apoptotic death of Phytochemistry 33, 657–661. human epidermoid carcinoma A431 cells via caspase Das, S., Das, S., Mujib, A., Pal, S. and Dey, S. (1998) activation together with dissipation of mitochondrial Optimisation of sucrose and dissolve oxygen level for membrane potential and cytochrome c release. somatic embryo production of Santalum album in airlift Carcinogenesis 26, 369–380. bioreactor. Prensa Aromatica 14,12–13. Kokate, C.K., Purohit, A.P. and Gokhale, S.B. (1998) Dastur, J.F. (1962) Medicinal Plantsa of India and Pakistan. Pharmacognosy, 23rd edn. pp. 106–114. Pune: Nirali Bombay: DB Taraporevala Sons and Co. Pvt. Ltd. Prakashan.

Koricheva, J., Larsson, S., Haukioja, E. and Keinaenen, M. Shankaranarayana, K.H. and Kamala, B.S. (1989) Fragrant (1998) Regulation of woody plant secondary metabolism products from less odorous sandal oil. Perfu Flavorist 14, by resource availability hypothesis testing by means of 19–20. meta-analysis. Oikos 83, 212–226. Shankaranarayana, K.H. and Parthasarathi, K. (1987) On the Kumar, G.M., Jeyraaj, I.A., Jeyaraaj, R. and Loganathan, P. content and composition of oil from heartwood at (2006) Antimicrobial activity of aqueous extract of leaf different levels in sandal. Indian Perfumer 31, 211–214. and stem extract of Santalum album. Anc Sci Life 25,3–4. Sikkema, J., De Bonte, J.A.M. and Poolman, B. (1995) Lacey, R.W. and Lord, V.L. (1981) Sensitivity of Staphylococci Mechanisms of membrane toxicity of hydrocarbons. to fatty acids: novel inactivation of linolenic acid by Microbiol Rev 59, 201–222. serum. J Med Microbiol 14, 41. Simanjuntak, P. (2003) Antibacterial assay of sandalwood Lloyd, G. and McCown, B. (1981) Commercially-feasible (Santalum album L.) extract. Majalah Farm 14, micropropagation of Mountain laurel, Kalmia latifolia, 326–332. by use of shoot tip culture. Int Plant Prop Soc Proc 30, Skaltsa, H.D., Demetzos, C., Lazari, D. and Sokovic, M. (2003) 421–427. Essential oil analysis and antimicrobial activity of eight Maier, W., Peipp, H., Schmidt, J., Wray, V. and Strack, D. Stachys from Greece. Phytochemistry 64, 743–752. (1995) Levels of a terpenoid glycoside (blumenin) and Takaishi, Y., Ochi, T., Shibata, H., Higuti, T., Kodama, K.H. cell wall-bound phenolics in some cereal mycorrhizas. and Kusumi, T. (2005) Anti-Helicobacter pylori compounds Plant Physiol 109, 465–470. from Santalum album. J Nat Prod 68, 819–824. Mondello, F., de Bernardis, F., Girolamo, A., Cassone, A. and Tanaka, N., Shimomura, K. and Ishimaru, K. (1995) Tannin Salvatore, G. (2006) In vivo activity of terpinen-4-ol, the production in callus cultures of Quercus acutissima. main bioactive component of Melaleuca alternifolia Cheel Phytochemistry 40, 1151–1154. (tea tree) oil against azole-susceptible and -resistant human Vatai, T., Skerget, M., Knez, Z., Kareth, S., Wehowski, M. and pathogenic Candida species. BMC Infect Dis 6, 158. Weidner, E. (2008) Extraction and formulation of NCCLS (2000) Methods for dilution antimicrobial anthocyanin- concentrates from grape residues. J Supercrit susceptibility tests for bacteria that grow aerobically. In Fluids 45,32–36. Approved Standard, 5th edn. NCCLS document M7-A5. Viollon et Chaumont. (1994) Parfums and Cosmetiques 116, Wayne: NCCLS. 67–70. Nostro, A., Germano, M.P., Angelo, V.D., Marino, A. and Wiermann, R. (1981) The Biochemistry of Plants ed. Cannatelli, M.A. (2000) Extraction methods and Stumpfand, P.K. and Conn E.E., Vol. 7, pp. 85–116. New bioautography for evaluation of medicinal plant York: Academic Press. antimicrobial activity. Lett Appl Microbiol 30, 379–384. Winter, A.G. (1958) Significance of volatile oils for treatment Okasaki, K. and Oshima, S. (1953) Antimicrobial effect of of urinary passage infections. Planta Med 6, 306. essential oils. J Pharm Soc 73, 344. Yamashita, Y. (1997) Production of essential oils by culture of Patil, V., Vadnere, G.P. and Patel, N. (2011) Absence of the callus of sandalwood tree. Patent No: JP09023892. antimicrobial activity in alcoholic extract of Santalum album Linn. J Pharm Negat Results 2, 107–109. Rangasamy, O., Raoelison, G., Rakotoniriana, F.E., Cheuk, K., Supporting Information Urverg-Ratsimamanga, S., Quetin-Leclercq, J., Gurib- Additional Supporting Information may be found in the Fakim, A. and Subratty, A.H. (2007) Screening for anti- online version of this article: infective properties of several medicinal plants of the Mauritians flora. J Ethnopharmacol 109, 331–337. Figure S1. Percentage of phytochemicals in sandalwood Rangaswamy, N.S. and Rao, P.S. (1963) Experimental studies in vitro (a, b, c) and in vivo (d, e) extracts. The colour on Santalum album L. – establishment of tissue culture guide corresponding to major groups of metabolites is of endosperm. Phytomorphology 13, 450–454. provided (f). The values mentioned alongside the metabo- Rao, B. (2003) Bioactive phytochemicals in Indian foods and lite groups are indexed as a relative value to each other their potential in health promotion and disease and the sum of the extract quantified. – prevention. Asia Pac J Clin Nutr 12,9 22. Table S1. Physicochemical and phytochemical charac- Riipi, M., Haukioja, E., Lempa, K., Ossipov, V., Ossipova, S. terization of extracts. and Pihlaja, K. (2004) Ranking of individual mountain birch trees in terms of leaf chemistry: seasonal and annual Please note: Wiley-Blackwell is not responsible for the variation. Chemoecology 14,31–43. content or functionality of any supporting materials sup- Rodriguez Vaquero, M.J., Alberto, M.R. and de Nadra, M.C.M. plied by the authors. Any queries (other than missing (2009) Antibacterial effect of phenolic compounds from material) should be directed to the corresponding author different wines. Food Control 18,93–101. for the article.