Initial Evidence of Plant and Animal-Origin Organic Additives from the Second-Century Bce Earthen Plaster of Rock-Cut Caves of Karla, India**

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Archaeometry ••, •• (2019) ••–•• doi: 10.1111/arcm.12522

INITIAL EVIDENCE OF PLANT AND ANIMAL-ORIGIN ORGANIC ADDITIVES FROM THE SECOND-CENTURY BCE EARTHEN PLASTER OF ROCK-CUT CAVES OF KARLA, INDIA**

B. DIGHE and M. R. SINGH† Department of Conservation, National Museum Institute, Janpath, Delhi, 110011, India

Analytical investigations through phytochemical screening, scanning electron microscope (SEM) and light microscopic observation of the earthen plaster of Karla Caves of western India identified the presence of antifungal, antibacterial and insect repellent Careya arborea stem fibres as a vegetal additive. The scanning electron microscopy with energy-dispersive X-ray spectrometry (SEM-EDX) study also revealed the inclusion of rice (Oryza sativa) husk as a plant additive in the plaster. In the high rain-fed coastal regions of western India, the antimicrobial, antioxidant and termite-resistance properties of C. arborea helped the survival of the plaster in unfavourable climatic conditions. Besides, the C. arborea and rice husk together played a role in reducing cracks, decreasing the density, and imparting a plasticity and a durability to the plaster. The liquid Fourier-transform infrared spectrometer (FTIR) of organic extracts revealed carboxylic acid (fatty acids)-based additives in the earthen plaster. From the gas chromatography-mass spectrometry (GC-MS) analysis of the plaster, the presence of animal milk fat, animal and vegetable fat, and vegetable oil was identified, and probably used to strengthen the earthen plaster for their resistance to tensile stress. The analysis also diag- noses the inclusion of methyl commate B, a resinous material obtained from the Burseraceae plant family, which has antimicrobial properties, through GC-MS analysis.

KEYWORDS: ORGANIC ADDITIVES, ANTIMICROBIAL, EARTHEN PLASTER, CAREYA FIBRES, GC-MS, MILK FAT

INTRODUCTION An interest in earthen construction has re-emerged due to the current environmental scenario around the world. Although cement has replaced ancient binders such as lime, mud and gypsum, 50% of the world’s population still lives in earthen dwellings (Kabiraj and Mandal 2012). Earthen buildings are preserved over the centuries by resisting harsh weather conditions, and therefore it is essential to understand the ancestral practice of earthen construction. In western India, many of the decorative arts pertaining to between the second century BCE and the tenth century CE have been executed on earthen plaster supports. These historical plasters are now showing deterioration and are in need of urgent restoration. To design an appropriate methodol- ogy, wall painting conservators are supposed to be familiar with the conservation problems of the earthen support layer. Therefore, the identiﬁcation and characterization of each constituent of the earthen plaster is vital for a proper understanding of the original fabric of the earthen support. Such a characterization will allow the development of novel materials whose composition can be matched with the original. Moreover, the support system for decorative art is prepared by ap- plying several earthen plaster layers of varied composition and ﬁnished with a thin lime wash

*Received 20 December 2018; accepted 12 November 2019 †Corresponding author: email [email protected]

© 2019 University of Oxford, Archaeometry ••, •• (2019) ••–•• 2 B. Dighe and M. R. Singh layer that will receive the colours. In the complex system of India’s Ellora caves (Singh et al. 2015), the support has been prepared by the application of successive lime and earthen plaster layers of distinct character. Earthen mortars are mainly prepared by mixing sand, silt and clay, and for optimal performance of the earthen support, it is desirable to have the correct distribution of these components. In order to overcome the in-adoptability of local raw materials, other organic and inorganic additives are often mixed in the plasterwork. The additives mixed in the earthen plaster significantly influence its shrinkage property, besides acting as cementing agent. If the clay content of the sourced soil is > 60%, the compactness of the mixture of plaster will increase (Hashmi 2008). Further, expandable clays will lead to a greater compactness than the non-expandable variety which leads to the very effective binding of sand and silt particles in the plaster. In order to improve the impermeability of water and to enhance the durability of the plaster, several biopolymers in the form of fats and oils are found to be added to the plaster (Eires et al. 2015). The earthen plaster prepared using larger grain particles is prone to damage by grain breakage on even slight impact, and organic additives derived from plants and animals are found to be mixed pref- erentially in these plaster to act as a cementing agent. The ancient earthen plasters are also found to be added with vegetal additives such as straw, jute and rice husk to prevent excessive cracks during drying. The earthen plaster consisting of a high aggregate-to-binder ratio (the addition of additives when mixed with mud) improves the physical properties and performance of the plaster. There are two sources for organic materials: vegetal, for example, straw, palm leaves, rice husk, hemp etc.; and animal, such as animal hair and dung (Torraca 1982). The additives should be water resistant, should not close the pores and capillaries of the plaster, and should have good penetration. They must be durable, easily ap- plicable, cheap, and should not be chemically hazardous (Ahmadi 2008). The amount of vegetal additive mixed in the soil varies from case to case based on the type of additives and soil composition. The fibres found mixed in Indian plasterwork are mainly made from plant cell walls and mostly composed of carbohydrates and other substances. These fibres reduce the tensile strength, reduce cracking, decrease density and speed up the drying of the plaster. The improvement in the characteristics of the plaster varies according to the size, shape, strength, quantity, elasticity and bond strength of the fibres with soil particles. Commonly used fibres are those derived from wheat, rice, barley or their chaff or husk. In addition, other vegetal fibres, such as hey, hemp, millet, sisal, elephant grass etc., have been used. One negative impact of using vegetal fibres is that they do not increase the comprehensive strength of the plaster. The vegetal additives also have an inconsistent durability. The dry fibres will survive for a long period, but wet fibres are liable to rot or may be attacked by insects, termites, etc. (Berhane 1990; Abdu 2010). However, for the earthen plaster in a higher rainfed area, the vegetal fibres selected are those that have antifungal, antibacterial and insect repellent properties. One such example is the Buddhist mural art in the rock-cut caves at Karla in India’s Western Ghats, about 50 km west of Pune. India’s Western Ghats are known for their dense forest and high rainfall, leading to an increase in humidity inside the caves up to 90% (Singh et al. 2016). The large size, open and damaged doors do not regulate the humidity inside the caves. The high clay (70%) and low silt/sand soil sourced for the plaster may lead to insect activity and fungal attack, leading to the loss of painted plaster. However, there is a selective use of plant additives by the ancient technicians to undo the cause for the loss of the plaster. There are several examples of the addition of biopolymers in earthen buildings in order to improve their impermeability to water and material durability (Chang et al. 2015). Proteins, poly- saccharides, nucleic acids and carbohydrates are examples of biopolymers of natural or

© 2019 University of Oxford, Archaeometry ••, •• (2019) ••–•• Earthen plaster organic additives of Karla caves 3 biological origin. The vegetal additives, such as ﬂour, starches, gum, oils, waxes or plant resins, or animal-origin biopolymers, such as fats, casein, egg white, blood, excrement and urine, were found to be added to earthen plaster (Fang et al. 2014). The exact identiﬁcation of these proteinaceous materials is not easy as they have altered much over the course of time. The aim of the present study is to analyse the vegetal additives and biopolymers mixed in the earthen plasters of Karla Caves in order to prepare compatible restoration materials. The components responsible for enhancing properties leading to resistance of the plaster to biocorrosion and termite resistance are also investigated.

MATERIALS AND METHODS

Studied site

Karla Caves (second century BCE) is located about 50 km west of the historical city of Pune, 18.7833°N, 73.4704°E (Fig. 1, a). The caves were excavated through living basalt rock and played a very important role in maritime trade between 100 and 300 BCE (Fig. 1, b). The caves are a good example of the earliest excavation and earthen plaster technology for the decorative arts of Buddhist origin in western India (Fig. 1, c). The decorations were done in several stages.

Figure 1 (a) Location of Karla Caves, western India; (b) general view of Karla Caves; (c) Grand Chaitya from Cave no. 8; and (d) earthen plaster admixed with organic additives on the rough stone surface. [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

First, the stone surface was chiselled to make a rough surface onto which the plaster admixed with clay, lime and organic additives was laid (Fig. 1, d). A lime wash layer was applied on the dried plaster surface to receive the colour. The caves have been researched by various archaeologists/art historians, and based on the archaeological evidence, the caves are dated to the second century BCE (Burgess 2013). In order to identify the plant ﬁbres, husk and organic additives admixed in the earthen plaster, scientiﬁc investigations such as phytochemical screening, scanning electron microscopy coupled with energy-dispersive X-ray spectrometry (SEM-EDX), liquid Fourier-transform infrared spectrometer (FTIR) and gas chromatography-mass spectrometry (GC-MS) were carried.

Phytochemical screening of the fibres Phytochemical screening of the methanolic extract of the isolated fibres and the collected reference materials for the presence or absence of secondary metabolites such as saponin, flavonoids, tannin, terpenes and alkaloids was carried using various chemical assays (Horborne 1998). For the collection of reference materials, a survey was conducted in the surrounding reserved forest, where the tribal population extensively uses plant stem fibres obtained from sisal (Agave sisalana), flax (Linum usitatissimum) and kumbhi (Careya arborea) for domestic purposes. The phytochemical test includes: • Determination of saponin: methanolic extract dissolved in water is shaken vigorously. There is a honeycomb-shaped persistent froth. A persistent foam indicates the presence of saponin. • Determination of flavonoids: 1.0 ml extract plus a few drops of concentrated HCl + magne- esium. The development of a pink colour indicates the presence of flavonoids. • Determination of tannin: 1.0 ml extract + 1.0 ml 5% FeCl3. The development of a dark green colour indicates the presence of tannin. • Determination of terpenes: 1.0 ml extract + 5.0 ml chloroform + 2.0 ml conc. H2SO4. The development of a reddish brown colour at the interface indicates the presence of terpenes. • Determination of alkaloids: 1.0 ml extract was dissolved in 2.0 ml of 5% HCl; after mixing, three aliquots were taken. Drops of Wagner, Mayer and Dragendorff reagents were added to each. The red-brown precipitate (Wagner), the yellowish-white precipitate (Mayer) and the red-orange precipitate (Dragendorff) indicate the presence of alkaloids.

SEM observation of the fibres The thin sections of fibres were treated with a 1:1 mixture of 99% glacial acetic acid (CH3COOH) and 50% hydrogen peroxide (H2O2) for 2 h at 60°C so as to remove the attached earthen plaster, followed by cleaning with a soft brush. Cleaning was performed until there were no traces of earthen plaster. The sections were observed under a light microscope and SEM. For SEM, the sections were placed on a stub and sputter coated with gold (Au) using an SPI- MODULE sputter coater and observed under a Zeiss model SmartSEM at the Central Research Facilities, IIT, Delhi. The qualitative chemical tests and microscopic observations for the anatomical characters of plant fibres (reference materials) and isolated plant fibres from the plaster were carried in order to test the distinctive chemical and anatomical characters. This included the chemical test for the presence of different secondary metabolites and anatomical characters such as the types of vessel ray pits, inter-vessel pits diameter and presence of prismatic crystals.

Husk sample analysis

The husk samples were treated with a 1:1 mixture of 99% glacial acetic acid (CH3COOH) and 50% hydrogen peroxide (H2O2) for 2 h at 60°C so as to remove the attached earthen plaster, followed by cleaning with a soft brush. Subsequently, the sample was observed under a light microscope and SEM in order to observe its morphological characters and the results were compared with a fresh paddy husk sample (reference material), while Energy Dispersive X-ray Spectrometry (EDX) was performed for its chemical characterization.

FTIR analysis In order to extract non-polar and polar components, the samples were prepared using different organic solvents such as hexane, toluene and ethyl acetate (all Sigma-Aldrich) (Rampazzi et al. 2016). A total of 50 mg of the ﬁnely powdered samples was placed in a glass test tube, con- taining organic solvents (around 0.5 ml) and sonicated for 15 min and then subsequently centri- fuged for 5 min. The soluble fraction was placed on a NaCl plate and analysed in transmission mode after gentle evaporation of the solvent. Similarly, extraction with deionized water was also carried. The residue was analysed as KBr pellets using a Bruker Vertex 70v FTIR spectrometer in transmission mode (400–4000 cmÀ1) at the Centre for Nanoscience and Nanotechnology, Jamia Millia Islamia University, Delhi.

GC-MS analysis for biopolymers The earthen plaster sample was characterized for the presence of biopolymers (Krizova et al. 2018). The GC-MS analysis of the earthen plaster was carried out at the Advanced Instrumenta- tion Research Facilities, JNU, Delhi. The selected homogenized earthen plaster sample (100 mg) was dissolved in 100 ml of methanol (HPLC grade; Sigma-Aldrich) at laboratory temperature for 24 h. The sample was then filtered using Whatman filter paper (Grade 1). The filter samples were analysed using a GCMS-QP2010 Ultra instrument (Shimadzu, Kyoto, Japan), equipped with an AOC-5000 plus autosampler, an OPTIC-4 injection system and a non-polar Restek Rxi-5 Sil MS column (5% diphenyl–95% dimethylpolysiloxane, 30 m × 0.25 mm i.d., × 0.25 μm film thick- ness). Helium (99.999% Linde) was used as the carrier gas at a flow rate of 0.9 ml min–1. A split injection mode at 260°C with a split ration of 10.0 and sample volume of 1.5 μl was employed. The column temperature was held at 60°C for 2 min and ramped at 10°C min–1 to 280°C for 18 min (the total run time was 40 min). The MS was operated with electron impact ionization (70 eV) with full scan monitoring. Interpretation of the GC-MS chromatogram was conducted using a database of the National Institute Standard and Technology (NIST) and Wiley having > 62 000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in either library.

RESULTS AND DISCUSSION Small chopped fragments of the fibres and husks were found mixed in the earthen plaster of Karla Caves. The chopped fibres were seem to be obtained from the plant stem. Few fibres appeared to be thick walled, showing the presence of well-preserved secondary wood structures.

Plant fibres and husk identification The plant fibre and husk inclusion in the earthen plaster were identified to be the stem fibres of C. arborea and rice (O. sativa) husk, which was intentionally added to the plaster so as to avoid cracks and impart plasticity to the plaster.

0.0.1. Plant fibre identification In relation to the above study, the features found most useful for a correct identification were as follows: • The phytochemical screening of the methanolic extract of reference materials and the isolated plant fibres from the earthen plaster for the presence of secondary metabolites are shown in Table 1. The phytochemical test of the isolated plant fibres showed similar chemical properties with the the freshly collected C. arborea stem fibres. Hence, the freshly collected C. arborea fibres were further used for anatomical studies. • Two types of vessels’ ray pits were present, which are observed to be often different ray cells (Lens et al. 2007). (1) Circular to oval bordered pits with reduced and simple borders, horizon- tally elongated. The diameter of the pit cavities was about 10–12 μm (Fig. 2, a, b). (2) Crowded pits, their border assuming to be in rectangular outlines and arranged in diagonal rows, with a 6 μm cavity diameter (Fig. 2, c, d). Two fundamental types of tracheary elements occur in the xylem: tracheids; and vessel elements or vessel members. The tracheary elements are con- nected through openings in the lignified secondary walls known as pits (Evert 2006). The pri- mary walls and intervening middle lamella of two opposite cells constitute the pit membrane, which lies in the centre of the pit pair. Water and nutrients are transported between vessels through bordered pits in the xylem tissue of hardwood species (Jansen et al. 2009). The func- tioning of the xylem network is largely dependent on bordered pits that connect adjacent con- duits. The bordered pits in tracheary elements show three main types of arrangement: opposite, alternate and scalariform. Circular or oval bordered pits arranged in horizontal pairs or short horizontal rows characterize opposite pitting. If such pits are crowded, their borders assume rectangular outlines in a face view; when the pits are arranged in diagonal rows, this arrangement is alternate pitting. If the pits are elongated transversely and arranged in vertical, ladder- like series, the pattern is called scalariform pitting (Evert 2006). • The presence of tyloses with prismatic crystals (Fig. 3). The tyloses most commonly occur in the pitted vessels of the metaxylem and secondary xylem and they are rarely found in protoxy- lem elements with an annular or spiral secondary wall (De Micco et al. 2016). They are also found occasionally in the tracheids of both soft- and hardwoods and in the fibres of hardwoods (Chrysler 1908; Esau 1965). These types of vessel occlusion play a crucial role at limiting the spread of pathogens and wood decay organisms (De Micco et al. 2016).

Table 1 Phytochemical screening of the secondary metabolites present in reference materials and isolated plant ﬁbre

Methanolic extract Saponin Tannin Terpene Flavonoids Alkaloids Sisal fibres ++ ++ À ++ À Careya fibres ++ +++ + ++ + Flax fibres ÀÀ +++ Isolated plant fibres + ++ + + +

+++, Strong intensity reaction; ++, medium intensity reaction; +, low intensity reaction; À, not detected.

Figure 2 Scanning electron micrographs (SEM) of two types of vessels ray pits: (a, b) circular bordered pits of new (reference material) and old Careya arborea fibre; and (c, d) crowded bordered pits of new (reference material) and old C. arborea fibre. [Colour figure can be viewed at wileyonlinelibrary.com]

• Two types of ray cells present: uni- and multiseriate. Uniseriate rays were found to be infre- quent, while multiseriate rays were frequent with two to seven serrate, consisting of a mixed procumbent and square body ray cells (Fig. 4). The ray parenchyma cells of the angiosperms vary in shape, but two fundamental forms may be distinguished: procumbent (having their longest axes oriented radially) and upright ray cells (having their longest axes oriented vertically). In angiosperms, rays composed of one kind of cell are called homocellular, and those contain- ing procumbent and upright cells are heterocellular. In comparison with predominantly uniseriate rays of conifers, those of the angiosperms may be one to many cells wide, that is, they may be uni- or multiseriate. The rays of angiosperms average about 17% of the volume of the wood, contributing substantially to its radial strength (Evert 2006). • Prismatic crystals were found to be common in the body and marginal ray cells (Fig. 5, a, b). Such crystals in plants are formed from an endogenously synthesized oxalic acid and Ca from the environment (Franceschi and Nakata 2005). Antonie van Leeuwenhoek in late 1600s was the ﬁrst to describe calcium oxalate crystals in plants using a simple light microscope (Arnott and Pautard 1970). The major function of prismatic crystals in plants includes Ca regulation, plant protection against herbivores and metal detoxiﬁcation (Franceschi and Nakata 2005).

Figure 3 Light microscopic image showing tyloses in the Careya arborea ﬁbre. [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

Figure 4 Scanning electron micrograph (SEM) of uni- and multiseriate rays of Careya arborea ﬁbres. [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

On the basis of these features, the ﬁbre was assigned to the Lecythidaceae family belonging to genus Careya and species arborea, having the same studied features. C. arborea, commonly known as wild guava, is widely distributed in India, Sri Lanka and the Malay Peninsula (Gupta et al. 2012). The plant is very important in medicine for the treatment of tumours, bronchitis, skin diseases, epileptic ﬁts, astringents, abscesses, boil, ulcers, and an antidote to snake venom

Figure 5 Scanning electron micrographs (SEM) of prismatic crystals of Careya arborea fibre from (a) a new (reference material) C. arborea; and (b) an isolated plant fibre sample. [Colour figure can be viewed at wileyonlinelibrary.com]

(Nadkararni 2004). The leaves and ﬂowers in the form of paste are used to cure skin diseases, diarrhoea, dysentery and ear pain (Gupta et al. 2012). C. arborea is reported to possess cytotoxic activity (Senthilkumar et al. 2007), antitumor effects (Natesan et al. 2007), is a central nervous system depressant (Kumar et al. 2008) and an anticoagulant (Varadharajan et al. 2010). Being a medicinally important plant, it also possess antibacterial and antifungal activities. The methanol extract of the bark of C. arborea was found to be effective against certain bacterias such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typi etc., and also against certain fungus species such as Candida albicans, Aspergillus ﬂavus and Alternaria solani, etc. (Sambath Kumar et al. 2006). C. arborea also possess antioxidant activity, which counteracts the deterioration of stored food products. Therefore, antibacterial, antifungal and antioxidant activities in C. arborea suggest it was added intentionally to the earthen plaster support so as to hinder bacterial and fungal growth in the plaster and to make its survival lifelong in the high rain-fed areas of India’s Western Ghats.

Husk identification • The exterior surface had the dentate rectangular elements which themselves are composed mostly of silica coated with a thick cuticle and surface hairs (Fig. 6, a, b). • The EDX data confirm the presence of amorphous silica in the dentate rectangular elements, concentrated on the surface of the rice husk. No other plant except paddy husk can retain such a huge amount of silica within it (Fig. 7). On the basis of these features, the husk was attributed to the Poaceae family belonging to genus Oryza and species sativa. The chemical composition of rice husk contains cellulose 40–50%, lignin 25–30%, ash 15–20% and moisture 8–15% (Igwebike-Ossi 2017). Paddy rice (O. sativa) is grown in every continent, except Antarctica, and the extent of paddy cultivation covers about 1% of the Earth’s surface (Maclean 2002). Rice production is dominated by Asia, where rice is the only food crop that can be grown in the rainy season. India and China account for over half the world’s rice supply (Muthayya et al. 2014). The rice husk is a potential material, responsive for valuable additions. The use of rice husk is found in many fields. Rice contains large amount of silica, which is important for normal growth and plant development. Silica is commonly accu- mulated in specialized short epidermal cells (silica cells) in which it is deposited initially in the

Figure 6 Scanning electron micrograph (SEM) of the morphological characters of rice (Oryza sativa) husk: (a) a new sample; and (b) an old sample. [Colour ﬁgure can be viewed at wileyonlinelibrary.com] cell wall and then accumulates centripetally. Silica deposition protects the plant from bacterial and fungal pathogens, supports the stems, reduces the uptake of toxic metals and regulates water loss (Isa et al. 2010), and also strengthens the earthen plaster.

Investigation of the organic fraction by FTIR The extracted organic samples with various organic solvents were subjected to FTIR analysis. As no absorption peaks were obtained from the sample extracted using hexane and toluene, the spectra recorded by ethyl acetate extraction are shown in Figure 8. The broad band at 3265 cmÀ1 observed represents the O-H band of alcohol and phenols present in the sample; the broad band at 2565 cmÀ1 denotes carboxylic acid (Merlic and Strouse 1997); the stretching absorption band at 1458 cmÀ1 represents amide bands from proteinaceous additives; the stretching absorbance bands of O-C at 1398 and 1113 cmÀ1 denote a carboxylic acid derivative; the peaks at 954 and 486 cmÀ1 represent silica; and the small band around 730 cmÀ1 may represent traces of calcite (Shearer 1989). The FTIR peaks suggest that the sample obtained after extraction with ethyl acetate might contain a fatty acid. The strong bands around 3265 and 2565 cmÀ1 ascribed to O-H group stretching and around 1113 cmÀ1 due to O-C bands are particular features of fatty acids.

GC-MS analysis of the organic additive in the mud plaster The GC-MS chromatogram of the earthen plaster of Karla Caves showed organic additives sourced from different plant and animal origins and were added at the time of preparation of plasters, as shown in Figure 9. A total of three saturated fatty acids, two unsaturated fatty acids and five other components were identified. The retention time and area percentage of the identified compounds are shown in Table 2. The main saturated fatty acids identified were: myristic acid (14C), palmitic acid (16C) and stearic acid (18C). The main unsaturated fatty acids identified were oleic acid (18C) and linoleic acid (18C). The major constituents of fatty acids in animal milk fat are myristic acid, palmitic acid, stearic acid and monounsaturated fatty acid such as oleic acid and linoleic acid (Hermansen 1995). Hence, the GC-MS result suggests that animal milk fat was probably added as an additive while preparing the plaster. In addition, the other components

Figure 7 Energy-dispersive X-ray spectrometry (EDX) of an ancient rice husk showing a higher percentage of silica. [Colour figure can be viewed at wileyonlinelibrary.com] identified were methyl stearate, eicosenoic acid, squalene and oleate stigmasterol. Methyl stearate is a 19-carbon fatty acid methyl ester of stearic acid belonging to the class of triglyceride, found in combined form in natural animal and vegetable fats. Its presence suggests the addition of animal and vegetable fats as additives in the plaster. However, the presence of eicosenoic acid (a 20-carbon monounsaturated omega 9 long-chain fatty acid), stigmasterol (the plant sterol) and squalene, a linear triterpene synthesized in plants, indicate the use of vegetable oil as an organic additives in the plaster. The identification of methyl commate B indicates that a resinous material obtained from plant origin belonging to the Burseraceae family was also added as an organic

Figure 8 Fourier-transform infrared spectrometer (FTIR) peak of the organic additives extracted through ethyl acetate. [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

Figure 9 Gas chromatography-mass spectrometry (GC-MS) chromatograph of the organic additives in the mud plaster of Karla Caves.

Table 2 Gas chromatography-mass spectrometry (GC-MS) peaks of the organic compounds present in the mud plaster of Karla Caves

Peaks R. time (min) Area (%) Compound Source 1 20.23 0.45% Myristic acid Animal milk fat 2 22.66 16.48% Palmitic acid 3 23.63 2.06% Methyl stearate Animal and vegetable fat 4 24.62 8.00% Oleic acid Animal milk fat 5 24.70 1.84% Oleic acid 6 24.90 2.55% Steraic acid 7 25.57 6.87% Eicosenoic acid Vegetable oil 8 26.82 4.59% Eicosenoic acid 9 26.99 1.09% Oleic acid Animal milk fat 10 27.13 0.88% Eicosenoic acid Vegetable oil 11 32.99 1.71% Squalene Vegetable oil 12 36.71 2.34% Methyl commate B Plant resin additive in the plaster. The mixing of guggula resin obtained from Boswellia serrate belonging to the Burseraceae in the clay plasters is still practised in some parts of India (Agrawal and Pathak 2001). Resin drawn from numerous Burseraceae plants possess an antibacterial activity (Motlhanka et al. 2010).

CONCLUSIONS Natural organic fibres occur in luxurious abundance in many parts of the world. In the construction of earthen plaster and mortars, they have played a direct role in protection and energy saving. The findings help with the understanding that the people around the second century BCE were well aware of the characteristics of vegetal additives which were easily and readily available from the Earth’s resources. One of the major roles of fibres was in delaying and controlling the tensile cracking of the matrix, which reduced deformation at all stress levels and imparted a well-defined post-cracking and post-yield behaviour. However, the identification of C. arborea fibres and rice husk in the earthen plasters of Karla Caves signifies it as a kind of organic/inorganic hybrid plaster. From the GC-MS data, it was observed that vegetal and animal-origin biopolymers were added to the earthen plaster as cementing materials for durability and better performance. These types of composition in the earthen plasters has helped to improve its properties such as bonding, toughness, antifungal, antibacterial and antioxidant activities, as the natural fibres are biodegrad- able and sensitive to biological attack. The data will help in the preparation of compatible plaster for restoration.

ACKNOWLEDGEMENTS The authors thank Dr B. R. Mani, Vice-Chancellor of the National Museum Institute, Delhi, for his constant support and interest shown during the investigative studies; Dr Aditya Kanth, Anjali Sharma, Tahseen Karche and research staff at the National Museum Institute, Delhi, for technical support; Bhushan Shigwan, research scholar, at the Agharkar Research Institute, Pune, for help with the collection of reference plant materials; as well as Mr Ajai Kumar at the Advanced Instru- mentation Research Facilities, JNU Delhi, Mr Kuldeep Sharma at the Central Research Facility, IIT, Delhi, and staff members of the Centre for Nanoscience and Nanotechnology, Jamia Millia Islamia University, Delhi.

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