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Journal of Ethnopharmacology 142 (2012) 98104

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Scientic validation of the different purication steps involved in the


preparation of an Indian Ayurvedic medicine, Lauha bhasma
Balaji Krishnamachary a,b, Narendran Rajendran a,b, Brindha Pemiah a,b, Sridharan Krishnaswamy a, Uma
Maheswari Krishnan a,c, Swaminathan Sethuraman a,c, Rajan K. Sekar a,c,n
a
School of Chemical & Biotechnology, SASTRA University, Thanjavur 613 401, Tamil Nadu, India
b
Centre for Advanced Research in Indian System of Medicine, SASTRA University, Thanjavur 613 401, Tamil Nadu, India
c
Centre for Nanotechnology & Advanced Biomaterials, SASTRA University, Thanjavur 613 401, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Lauha bhasma (iron ash) is one of the iron-based herbo-metallic
Received 20 September 2011 preparations used in Ayurvedic medicine for treating various ailments due to iron deciency.
Received in revised form Materials and methods: The preparation of Lauha bhasma (iron ash) requires normal purication (heat
25 March 2012
treatment in vegetable and animal products), special purication (treatment with herbal constituents)
Accepted 10 April 2012
Available online 26 April 2012
and calcination steps aimed at converting the raw material to a suitable therapeutic form. In this study,
we have systematically and scientically evaluated through a series of qualitative tests and modern
Keywords: analytical tools the importance of the treating media.
Lauha bhasma (iron ash) Results: Our data demonstrates that these steps are necessary to remove the grease and scales in the
Ayurveda (knowledge of life)
raw material. While heating, microcracks appeared on the surface of the iron, which improved the
Herbo-metallic preparation
reactivity with the herbal constituents in addition to incorporating nanostructured features. Further,
Nanostructure
the use of plant products facilitated the removal of Fe3 present in the raw material by forming soluble
complexes. The Fe2 present in the raw materials also forms an insoluble complex with the herbal
constituents in the presence of UV radiation.
Conclusions: In conclusion, our data summarily suggest that the purication steps involved in the
preparation of Lauha bhasma (iron ash) are critical.
& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Iron plays an important role in oxygen sensing, electron


transfer and in catalysis, being a component of several metallo-
Ayurveda (knowledge of life) is the veda (knowledge) of Ayush proteins (Papanikolaou and Pantopoulos, 2005). The human body
(life) (Sharma, 2003). It is the science of life practiced in India for contains about 35 g of iron, which exists in two formsheme
centuries and many Ayurvedic practices predate written records iron (Fe2 ) and non-heme iron (Fe3 ) (Papanikolaou and
and were handed over to generations by word of mouth (Sharma, Pantopoulos, 2005; Chinsembu and Faul, 2010). Most of the iron
1994). Ayurvedic system adopts a holistic approach towards (Fe2 ) in hemeproteins is located in the center of the porphyrin
health care by balancing the physical, mental and spiritual ring structure (Lieberman et al., 2007). Lauha bhasma (iron ash) is
functions of the human body (Bhanu, 1997). Rasa Shastra (vedic an iron-based herbo-metallic preparation that has several ther-
chemistry) is one of the sections of Ayurveda (knowledge of life) apeutic applications. The Ayurvedic Formulary of India provides
that deals with herbo-mineral preparations called bhasmas (ash) methods of preparation of various bhasmas (ash). Accordingly, the
(Senthil Kumar et al., 2011). Rasayana (immunomodulation and preparation of Lauha bhasma (iron ash) involves two major steps:
anti-aging quality) and Yogavahi (ability to target drugs to the Sodhana (purication) and Marana (grinding and calcination). The
site) are characteristics of a properly made herbo-mineral pre- purication processes comprise samanya sodhana (normal puri-
paration, which is also nontoxic, readily absorbable, adaptable cation) and vishesha sodhana (special purication). During the
and assimilable in the body (Sarkar and Chaudhary, 2010). normal purication process, red-hot iron is treated sequentially
with sesame oil, buttermilk, cows urine, rice gruel and horse
gram decoction. In the special purication process, the treated
n
iron is further puried using herbal extracts. The puried iron is
Corresponding author at: Seshasayee Paper & Boards Chair Professor in
Chemical Engineering, School of Chemical & Biotechnology, SASTRA University,
then subjected to repeated calcination. These purication meth-
Thanjavur 613 401, India. Tel.: 91 4362 304270; fax: 91 4362 264120. ods were developed by practitioners of Ayurveda (knowledge of
E-mail address: ksrajan@chem.sastra.edu (R. K. Sekar). life) to detoxify the raw material by chemical transformations and

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.04.021
B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104 99

modify the properties of the therapeutic materials to enhance sodhana (special purication), bhanupaka (exposure to sunlight),
their potential. sthali paka (roasting in an iron pan) and putapaka (calcination). A
The traditional Ayurvedic practitioners have used various ow diagram depicting the steps in the preparation of Lauha
qualitative tests to characterize the bhasma (ash), which includes bhamsa (iron ash) is shown in Fig. 1.
varna (test for color), nishchandratvam (test for luster), varitara
(test for lightness), rekhapurnata (test for neness), niruttha (test
2.1.1. Samanya sodhana (normal purication)
for metallic form) (Anand and Neetu, 2010). Many variations in
The rst step in the preparation of Lauha bhasma (iron ash) is
the procedure for the preparation of Lauha bhasma (iron ash) have
sodhana (purication step), which involves sequential quenching
been reported in the literature (Singh and Reddy, 2011; Bhanu,
in tila thaila (sesame oil), takra (butter milk), kanjika (rice gruel),
1997). Hence it is imperative to understand the scientic basis of
gomuthra (cows urine), and kulatha kasaya (horse gram decoc-
various steps involved in the preparation of Lauha bhasma (iron
tion). As in a typical purication step, about 2 kg of the raw
ash). In this study, we have systematically and scientically
material (iron lings) was heated to red hot condition (  530
evaluated the importance of each of the purication steps
560 1C) and immersed in 2 L of quenching medium (room tem-
involved in the preparation of Lauha bhasma (iron ash).
perature) for 2 h. Each of the quenching processes was repeated
thrice with each treating liquid by using fresh medium every
time. These steps constitute samanya sodhana (normal purica-
2. Materials and methods
tion) and the Lauha (iron) obtained at this stage is called Suddha
Lauha (iron after normal purication).
2.1. Preparation of Lauha bhasma (iron ash)

The preparation of Lauha bhasma (iron ash) was carried out 2.1.2. Vishesha sodhana (special purication)
following the procedure described in the Ayurvedic Formulary of Coarse powders of dried fruits, viz., Phyllanthus emblica (Indian
India. The raw material (Iron lings) were procured from the gooseberry), Terminalia chebula (Chebulic myrobalans) and Termi-
market in Trichy, India and used as raw material for preparation nalia bellerica (Belleric myrobalans) were taken in equal quanti-
of Lauha bhasma (iron ash). The preparation involves the follow- ties (2 kg each) and mixed with 50 L of water. The resulting
ing major stepssamanya sodhana (normal purication), vishesha mixture was heated to reduce to one-eighth of the original

Fig. 1. Flow diagram showing various steps in the preparation of Lauha bhasma.
100 B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104

volume to obtain Triphala kasaya (decoction of three fruits). About cool to room temperature. When this process is repeated number of
2 kg of Suddha Lauha (iron after normal purication) was heated times, it is referred in the classical ayurveda texts as bhasmikarana
to red-hot condition and immersed in a mixture of 1 L each of (ashing). For the puta (calcination) process, the cakrikas (thin at
triphala kasaya (decoction of three fruits) and cows urine for 2 h. disks) were placed in an earthen vessel (Fig. 2(A)) and covered with
This purication step was repeated seven times using freshly another earthen vessel (Fig. 2(B)) and the interface between the
prepared mixture of triphala kasaya (decoction of three fruits) and vessels was sealed with a clay-smeared cloth (Fig. 2(C)). This
cows urine. The Lauha (iron) obtained at this stage is called Lauha arrangement is known as sarava samputa (sealed earthen pot).
after vishesha sodhana (iron after special purication). The calcination process in the preparation of Lauha bhasma (iron
ash) was carried out in a specially designed pit, measuring 90  90 
2.1.3. Bhanupaka (exposure to sunlight) 90 cm. Cow dung cakes each weighing about 350 g were used as heat
Triphala kasaya (decoction of three fruits) was added to Lauha source. The bottom half of the pit was stacked with several layers of
obtained after vishesha sodhana (iron after special purication) cow dung cakes, over which the sarava samputa (sealed earthen pot)
and allowed to dry under sunlight for 5 day. This process was containing cakrikas (thin at disks) were placed (Fig. 3). This was
repeated seven times and the Lauha at this stage is called Lauha followed by stacking several layers of cow dung cakes on top. The
after bhanupaka (puried iron after exposure to sunlight). number of cow dung cakes used in this process ranged between 250
and 300 and this arrangement is referred to as Gajaputa (calcination
in pit measuring one yard in three directions) (Fig. 3).
2.1.4. Sthalipaka (frying in iron pan)
Triphala kasaya (decoction of three fruits) was freshly prepared 2.2. Characterization
by adding 2.3 kg each of Indian gooseberry, Chebulic myrobalans
and Belleric myrobalans to about 110 L of water and reduced to In order to understand the stage-wise transformations in
one-eighth of its original volume. About 2.3 kg of Lauha after surface morphology, the solid material obtained after each
bhanupaka (puried iron after exposure to sunlight) was washed quenching step was characterized using a cold-eld emission
with water and placed in a sthali (iron pan), to which 13.5 L of scanning electron microscope (FE-SEM, JSM 6701F, JEOL, Japan).
Triphala kasaya (decoction of three fruits) was added and heated The crystallinity of the samples obtained after each purication
to dryness for about 1 h at 95100 1C. The solid material obtained step was analyzed using an X-ray diffractometer (D8 Focus,
after this purication process is referred to as Lauha after Bruker, Germany). The surface area of samples was determined
sthalipaka (puried iron after frying in iron pan). using BET isotherms obtained from a surface area analyzer (ASAP
2020, Micromeritics, USA). The infrared spectrum and the elec-
2.1.5. Puta paka (calcination) tronic spectrum were recorded using a FTIR analyzer (Perkin
About 670 g each of Indian gooseberry, Chebulic myrobalans Elmer, Germany) and UV-Visible Spectrometer (Lamda 750,
and Belleric myrobalans were added to 4 L of water and heated to Perkin Elmer, USA) with diffuse reectance accessory, respectively.
reduce to one-fourth of its original volume. This was added to
2 kg of Lauha after sthalipaka (puried iron after frying in iron 2.3. Potassium thiocyanate (KCNS) test for presence of Fe3
pan); triturated well and made into cakrikas (thin at disks),
which were dried and stored for the next transformation. About 1 mL of KCNS was added to 1 mL of sample to test for
In traditional literature, the process of puta (calcination) refers to the appearance of red color. Immediate appearance of red color
controlled heating of herbo-mineral preparations and allowing to indicates the presence of free iron in trivalent form. In the

Fig. 2. Arrangements for Sarava samputa [A] cakrikas in an earthen vessel; [B] earthen vessels before sealing and [C] interface sealed with clay-smeared cloth.

Fig. 3. Arrangements for Gajaputa [A] Sarava samputa over stacked cow dung cakes; [B] Sarava samputa sandwiched between layers of cow dung cakes.
B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104 101

absence of red color, few drops of concentrated nitric acid were oil before and after being used for purication. The spent sesame
added to decompose the complex and liberate free Fe3 and KCNS oil appeared dark in color due to transfer of greasy matter from
was added to test for the appearance of red color. iron. Fig. 5 shows the scanning electron micrographs of raw iron
and sesame oil-treated iron, demonstrating that the raw material
2.4. Photochemical reactor does not possess crystallites or grains in nanoscale ( o100 nm),
while several nanostructures were observed in the size range of
A photochemical reactor was designed to study the effect of UV
50100 nm for treated iron, which may be attributed to the
radiation during the process of bhanupaka (exposure to sunlight)
physical properties and temperature of the quenching medium.
(Fig. 4). A mixture of 0.4 g iron (III) chloride, 20 mL of triphala
High boiling liquids like vegetable or mineral oils are generally
kasaya (decoction of three fruits) and gallic acid (1 g) were placed
preferred over aqueous media for quenching due to the limita-
in a quartz tube and irradiated with 125 W UV source. After 2 h,
tions of the latter like inefcient heat transfer due to excessive air
the sample was checked for the presence of solid content.
bubbles formed on the surface. On the other hand, quenching in
cold oil renders more brittleness to the product than that in hot
3. Results and discussion oil due to the faster rate of cooling in the former case (Bhanu,
1997). In a similar trend, repetitive quenching (e.g., three times)
3.1. Heat treatment with sesame oil might induce more thermal stress causing cracks and fractures
thereby reducing the particle size.
Sesame oil is used to remove greasy matter from the raw
material. Table 1 summarizes the characteristics of fresh sesame
3.2. Heat treatment with buttermilk

After removing the hydrophobic impurities using sesame oil


treatment, oxide scales formed due to atmospheric oxidation of
the raw material, are removed by treatment with aqueous media
viz., buttermilk, rice gruel, cows urine and horse gram decoction,
which are traditionally known for this property. Accordingly, The
X-ray diffractograms of raw material, sesame oil-treated material
and buttermilk-treated material are shown in Fig. 6. The X-ray
diffractogram of the raw material showed a peak at a 2y value of
451, which indicates the presence of metallic iron. The sesame oil-
treated material showed an intense peak at 2y value of 361, which
indicates the presence of Fe2O3 (Fig. 6). The Fe2O3 content in the
buttermilk-treated samples had reduced, while the intensity of
the peak corresponding to metallic iron had increased appreciably
(Fig. 6). This conrms the hypothesis that buttermilk removes
rust (Fe2O3) formed due to oxidation on the surface of the raw
material. To conrm this further, the spent-buttermilk (after
treatment) was tested for the presence of Fe3 through KCNS
Fig. 4. Photochemical reactor used to study the inuence of UV radiation on test. Red color was not observed indicating the absence of free
reaction between FeCl3 and triphala kasaya. Fe3 in the spent-buttermilk. However, KCNS test when repeated

Table 1
Characteristics of fresh sesame oil and sesame oil after treatment.

Sample Appearance (qualitative) Solids content (qualitative) Plausible explanation

Fresh sesame oil Transparent, yellow colored Absent


Sesame oil after rst cycle of treatment Dark colored Yes Greasy matter transferred to oil; also dirt and ne rust
Sesame oil after second cycle of treatment Transparent, yellow colored Yes Removal of residual greasy matter
Sesame oil after third cycle of treatment Transparent, yellow colored Yes Removal of residual greasy matter

Fig. 5. Scanning electron micrographs of raw material and sesame oil-treated material.
102 B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104

Table 2
BET surface area of solid material after each treatment step.

Sample description BET surface area (m2/g)

Raw material 1.7061


Taila treated Lauha 2.6536
Gomutra treated Lauha 3.0384
Kulatha kasaya treated Lauha 3.9158

have also been observed due to these heat treatments (Fig. 7),
depending on parameters including chemical composition of
treating liquid, temporal variation of temperature and surface
energy.

3.6. Bhanupaka (exposure to sunlight) and sthalipaka


(frying in iron pan)
Fig. 6. X-ray diffractogram of raw material, intermediate after treatment with
sesame oil and buttermilk. During the process of bhanupaka (exposure to sunlight), Triphala
kasaya (decoction of three fruits) was added to Lauha after vishesha
after addition of concentrated HNO3 resulted in the appearance of sodhana (iron after special purication) and allowed to dry under
red color. This conrmed that Fe3 had formed a coordination sun, which is a slow process. Hence, more time is available for
compound with the constituents of the buttermilk and this reaction between the herbal constituents and Lauha after vishesha
complex was decomposed by addition of concentrated HNO3. sodhana (iron after special purication). This treatment procedure
is expected to maintain iron in Fe2 form without getting oxidized.
3.3. Heat treatment with rice gruel The sample originally in the form of a homogenous liquid, turned
into a suspension of solid particles after bhanupaka (exposure to
The buttermilk-treated material was heated to red-hot condi- sunlight). The solid particles are recognized as water-insoluble
tion and immersed in rice gruel. This process was repeated thrice, complexes of Fe2 with gallic acid and tannic acid present in
with fresh rice gruel being used every time. We observed that use Triphala kasaya (decoction of three fruits). Any Fe3 present in the
of rice-gruel (pH 2.2) is to remove of Fe3 through formation of sample forms a water-soluble complex with gallic acid and is
coordination compounds, which was conrmed by KCNS test. Rice carried away in the supernatant solution.
gruel contains high levels of phytic acid, a chelating agent and has Sthalipaka (frying in iron pan) is carried out by placing Lauha
been found to react with Fe2 or Fe3 salts at pH 3 giving rise to after bhanupaka (puried iron after exposure to sunlight) in an
iron complexes (Geisser, 1990). iron pan containing Triphala kasaya (decoction of three fruits) and
heated to dryness. Substantial changes in microstructure occurred
3.4. Heat treatment with cows urine during this process (Fig. 8). The intermediate obtained after
bhanupaka (exposure to sunlight) contains irregular shaped
The rice gruel-treated material was heated to red-hot condition nanostructures of 4050 nm in size, while those obtained after
and immersed in cows urine, which among many other constitu- sthalipaka (frying in iron pan) had spherical nanostructures in the
ents contains ammonia, uric acid and urea (Dutta et al., 2006; range of 2025 nm (Fig. 8). Thus during sthalipaka (frying in iron
Jarald et al., 2008). Ammoniated citric acid is known to dissolve pan), there is reduction in size and change in shape.
Fe2O3 and forms water soluble Fe2 and Fe3 complexes (Donald
and Irving, 1997). Uric acid is also known to form ureate-Fe3 and 3.7. Transformations during puta (calcination)
ureate-Fe2 complexes (Davies et al., 1986). Thus the presence of
ammonia and uric acid in cows urine results in formation of As per classical Ayurvedic practices, puta (calcination) process for
complexes with iron that are soluble in water and hence the scale the preparation of Lauha bhasma has to be repeated 60 times. The
(oxides of Fe3 ) is removed, which was conrmed by KCNS test. nal form of Lauha bhasma (iron ash) should contain Fe2 in the
form of a complex. The surface morphology, size and the presence of
3.5. Heat treatment with horse gram decoction organic moiety was evaluated after the 20th puta (calcination). Fig. 9
shows the surface morphology of the Lauha bhasma (iron ash)
The cows urine-treated material was heated to red-hot con- obtained after 20th puta (calcination). The surface shows the
dition and immersed in horse gram decoction, which contains presence of spherical particles with a diameter of about 17 nm.
gallic acid that forms water soluble complexes with Fe3 . Hence Fig. 10 shows FTIR spectrum of the Lauha bhasma (iron ash) obtained
removal of any remaining Fe3 in the raw material could be after 20th puta (calcination). The presence of bands between 2900 to
achieved upon treatment with horse gram decoction, which was 2800 cm  1 and 600 to 500 cm  1 indicates iron (II) in its chelated
conrmed by KCNS test. form. Electronic spectrum of the Lauha bhasma (iron ash) obtained
In each of the steps in samanya sodhana (normal purication), after 20th puta (calcination) in the solid state using diffuse reec-
progressive increase in surface area, probably due to microcracks tance accessory showed two bands in the visible region
formed during heat treatment is observed (Table 2), resulting in 15,430 cm  1 (3A1g to 3T1g transition) and 19,530 cm  1(3A1g to
3
increased uid-particle interactions (Rajan et al., 2007; Rajan T2g transition) conrming the presence of Fe2 in the complex
et al., 2008). During each step, when the red-hot material is (Lever, 1984). The presence of iron in the divalent state and as a
immersed in treating liquids, the rate of chelation of ligands complex along with the nanodimensionality aids in easy absorption
present in the liquids with Fe3 in the material increases. and greater bioavailability of the formulation.
Considerable changes in surface morphology, in terms of the size Dietary inhibitors destroy free Fe2 , while chelated iron is not
and shape of grains and/or crystallites (in sub-100 nm range), destroyed by inhibitors and is also absorbed to a greater extent
B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104 103

Fig. 7. Surface morphology of solid material after each treatment step during samanya sodhana.

Fig. 8. Surface morphology of Lauha after bhanupaka and Lauha after Sthalipaka.

Fig. 9. Scanning electron micrograph of Lauha obtained after 20th puta.

(Pineda and Ashmead, 2001). Chelated iron is less toxic than free 4. Conclusions
and hydrated metallic ions (Papanikolaou and Pantopoulos, 2005;
Heather et al., 2003). Hence, it is imperative to have iron in the The process of making Lauha bhasma (iron ash) as per the
divalent state to enter mucosal cells. Fe3 form is not preferred as Ayurvedic texts involves thorough and stringent purication steps
it has to be reduced to ferrous form in the intestinal lumens, which include the samanya sodhana (normal purication), vishe-
which is difcult. sha sodhana (special purication), bhanupaka (exposure to
104 B. Krishnamachary et al. / Journal of Ethnopharmacology 142 (2012) 98104

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