1

CHAPTER 1 – INTRODUCTION AND REVIEW OF LITERATURE

1.1 Ayurveda

Ayurveda originally conceptualized by the sages of ancient India, as the veda (science) of

ayush (life) is a traditional Indian system of medicine prevalent for 5000 years [1,2]. It is

the science of life practiced in India for centuries and many Ayurvedic practices predate

written records and were handed over across generations by word of mouth [3]. Ayurvedic

system adopts a holistic approach towards health care by balancing the physical, mental

and spiritual functions of the human body [4]. According to Ayurveda, the normal

homeostasis of human body is governed by three doshas (humours), namely vata (for

transport), pitta (for metabolism) and kapha (for storage). When the three doshas are in

equilibrium, biological systems exhibit normal functions in the body and any distortion or

disturbance or imbalance between these doshas may lead to a diseased condition [2,5,6].

The inception of practice of Ayurvedic system is evident from the ancient literature Caraka

samhita followed by other texts such as Susruta samhita, Astanga samgraha , Astanga

hridaya, Bhela samhita, Sharngadhara samhita and Bhavaprakasa (6thcentury BC to 16th

century AD). Various texts that existed over this entire period are a compendium of

information about all medical aspects. Caraka samhita provides complete information

about the internal medicine encompassing etiology, symptoms, treatment and medical care.

Susruta samhita deals with clinical surgeries including surgical diseases, diseases of ear

and eye. The classification of Ayurvedic medicine is exemplified in Astanga samgraha.

They are general medicines, pediatrics, diseases of special sense organs, mental diseases,

surgery, toxicology, gerontology and aphrodisiac. Sharngadhara samhita highlights the

pharmaceutics principles, while Bhavaprakasa reviews various new diseases and plant-

2

based drugs [7,8]. Ayurvedic system utilizes drugs of plant, animal and mineral origin .The

drugs can be either single or as compound formulations. Currently 1000 single drugs and

8000 compound formulations are in existence in Ayurvedic system. The compound

formulations are divided into two types viz. Kasthausadhi (primarily plant drugs) and

Rasaausadhi (primarily metals and minerals) [9]. Different plant-drugs based formulations

and metal-based formulations are listed in Table 1.1 [9,10].

Table 1.1 Different plant and metal based formulations in Ayurveda.

Name of the

formulation Description

Total

number of

formulations Asava & Arista Extracting the active principles in plant drugs by

fermentation process

37

Arka Liquid preparation obtained by distillation of plant drugs 4 Leha &Paka Semi-solid preparation of drugs 32 Kvatha curna Coarse powder of drugs to prepare kasaya 33 Guggulu Exudate obtained from plant Commiphora mukul as main

ingredient

12

Ghrta Ghee is boiled with prescribed drugs or decoction 44 Curna Fine powder of drugs 40 Taila Taila is boiled with prescribed drugs or decoction 62 Dravaka Liquid preparation by employing lavanas and ksaras 1 Ksara Alkaline substances obtained from the ash of drugs 13 Lepa Drugs in the form of paste for external applications 12 Vati & Gutika Drugs prepared in the form of pills or tablet 35 Varti, Netrabindu

& Anjana

Drugs used externally for eye as soft paste and as eye drop 8

Sattva Water extractable solid substance collected from a drug 1 Kupipakva

Rasayana

Drugs of metal and mineral origin. Heating was done by

subjecting valukyantra

10

Parpati Mercury based preparation 5 Pisti Triturating drugs with suitable liquids and exposed to

sunlight. Fire is not used for heating

4

Bhasma Drugs of metal and mineral origin by subjecting them into

various purification and calcination process

22

Rasayoga Drugs of mineral origin 55 Lauha Preparations containing Lauha bhasma as main

ingredient

12

3

1.2 Metal based medicines in Ayurveda

The use of metals and minerals for therapeutic applications has been in vogue for centuries

in the Ayurveda system. The concept of using metals as medicines was developed by

ancient Indians during various periods [11]. Rasasastra is a branch of Ayurveda dealing

with the alchemical and pharmaceutical process of metals and minerals of aquatic and soil

origin [12,13]. Ayurveda can be classified into three discrete periods viz. vaidika period,

samhitaa period and post-samhitaa period based on the prevalence of particular system of

management of health and disease. Vaidika period was the era of four vedas with emphasis

on healthy life style. In samhitaa and post-samhitaa periods, great Indian acharyas of

various era documented medicinal practices. The literature on therapeutics during samhitaa

period accentuated more on plant-based drugs rather than metal-based preparations. Even

though the usage of metals for therapeutic potentials began in the samhitaa period, bhasmas

(herbo-metallic preparations) came into existence in the post-samhitaa period.

Naagaarjuna, the father of Rasasastra lived during this period and the system came into

proper existence around 8th century where numerous scientific documentations were made

available [14].

Bhasmas are herbo-metallic preparations of any metal or mineral origin prepared in such

a way that the toxic nature of the metal or mineral are believed to be removed, while

enhancing its therapeutic property. The plant sources added during the preparation process

are believed to enhance the therapeutic potential of the drug. Several sequence of steps like

Shodhana, Bhavana, Jarana and Marana are involved in the preparation of bhasma. These

steps remove toxic effects and enhance the therapeutic value of these medicines [15].

Bhasma should be administered at appropriate therapeutic doses in a suitable vehicle for

4

the prescribed therapeutic indication. The different bhasma, their source, therapeutic

indications, vehicle and dosage are tabulated in Table 1.2 [9].

Table 1.2 Different types of bhasma, their source, therapeutic indications, vehicle

and dose.

Bhasma Source Therapeutic uses Vehicle Dose

(mg)

Abharaka

[16–18]

Mica Digestive impairment,

malabsorption syndrome, asthma,

bleeding disorder, cough, urinary

disorder, anemia, falling of hair,

skin disease, senility, impotency,

splenic diseases

Honey, ghee,

triphala kasaya,

ginger juice

125-

375

Kapardika

[19,20]

Calcium

(Cowrie

shell)

Digestive impairment, duodenal

ulcer, malabsorption syndrome,

tuberculosis, otorrhoea, diseases of

eye, disorders of blood, deficiency

of semen

Adhatoda juice,

lemon juice,

trikatu kasaya

250

Kasisa[21,22] Green

vitriol

Anemia, splenic disease, hiccup,

ulcer, erysipelas, dysmenorrhea,

poison, weakness, diseases of eye

Bakuci curna,

triphala kasaya,

honey

250

Godanti[23,24] Gypsum Digestive impairment, headache,

fever, cough, asthma

Honey, tulasi

juice, ghee, sugar

500

Tamra[25–27] Copper Diseases of abdomen, ascites,

inflammation, anemia, neurological

disease, poison, tuberculosis,

asthma, diseases of eye, skin

disorders, duodenal ulcer, pain,

fever, liver disease, splenic disease,

excessive flow of urine, phthisis,

digestive impairment, malabsorption

syndrome

Trikatu kasaya,

ghee, honey

31.25

-62.5

Trivanga[28–30] Lead,

Tin, Zinc

Diabetes mellitus, increased

frequency & turbidity of urine

Honey, butter 125

Naga[31-33] Lead Diarrhoea, malabsorption syndrome,

abdominal lump, piles, urinary

disorder

Turmeric juice,

jambhul juice

62.5-

125

Pravala [34] Calcium

(Coral)

Inflammation, excessive sweating,

cardiac fibrillation, osteoporosis,

dysuria, oligouria

Honey, satavari

kasaya

250

5

Mandura[35,36] Iron Splenomegaly, hepatomegaly,

jaundice, anemia, blood loss

Honey, triphala

kasaya

250-

500

Mukta[37] Pearl Cough, asthma, phthisis, chronic

fever, weakness of the heart, mental

disorder

Honey, butter,

milk

125

Muktasukti[38] Pearl Pain in the abdomen, fever, diseases

of blood, abdominal lump,

tastelessness, poison, digestive

impairment, weakness

Honey, lemon

juice

250-

500

Yasada [39,40] Zinc Malabsorption syndrome, excessive

sweating, tuberculosis, increased

frequency & turbidity of urine,

leucorrhoea

Honey, trikatu

kasaya

125

Rajata[41] Silver Cachexia, tissue wasting, increased

frequency & turbidity of urine,

alcoholism, poison, fever, splenic

disease, low intelligence, uterine

disorder

Honey, vettiver

kasaya

125

Lauha Iron anemia, jaundice, skin diseases,

diarrhea, pain, hyperacidity,

diseases of abdomen, helminthiasis,

disorder of adipose tissue, splenic

disease, inflammation and asthma

Honey, ghee,

trikatu curna,

triphala curna

120-

250

Vanga[42–44] Tin Cough, asthma, disorder of adipose

tissue, excessive sweating, phthisis,

anemia, helminthiasis, increased

frequency & turbidity of urine,

uterine prolapse, leucorrhoea,

menorrhagia, digestive impairment,

tastelessness, low intelligence, ulcer

Honey, ghee 125-

250

Vajra Mica Diseases of abdomen, ulcer,

disorder of adipose tissue, increased

frequency & turbidity of urine,

inflammation, tuberculosis, diseases

of eye

Honey 8

Viakranta[45] Tourmali

ne

Malabsorption syndrome, anemia,

cough, asthma, piles, chest wound,

tuberculosis, worm infection

Honey, ghee 62.5

Sankha[46–48] Calcium

carbonate

Dyspepsia, digestive impairment,

malabsorption syndrome,

hyperacidity, duodenal ulcer, hepato

splenomegaly, poison

Lemon juice,

triphala kasaya

250-

300

Srnga Horn of

deer

Hiccup, cough, asthma, pleural

effusion, angina pectoris

Ghee, honey 250-

500

6

1.3 Methods adopted for preparation of Bhasma:

In general the metals or minerals are coarsely powdered and subjected to sodhana

(purification process), bhavana (levigation) and marana (calcination process). In sodhana,

a particular metal is heated to red hot condition or melted and quenched in particular liquid

media for specified number of times. The purified materials are then mixed with specific

drugs for incineration (maraka dravyas) and are levigated (bhavana) using particular liquid

media for a specific time period. Bhavana is a process of wet grinding, in which materials

are ground with particular liquid media for a specific period.

Cakrikas (pellets) are prepared from levigated doughy mass and placed in one earthen

plate, covered with another and the junction sealed by mud smeared cloth. The apparatus

(sarava samputa) is subjected to heating in traditional puta (calcination). The process of

heating of materials in the pit (made of specific dimensions) using cow dung cakes is called

as putapaka. Burning is continued for a specific period of time. The earthen plates (sarava

samputa) are taken out after being cooled naturally and opened to recover incinerated

Svarna [49,50] Gold Malabsorption syndrome, hyper

acidity, hiccup, anemia, asthma,

fever, tissue wasting, tuberculosis,

impaired intelligence, epilepsy,

weakness of ligaments, heart

diseases, loss of body complexion,

syphilis, poison, hoarseness of

voice, aphrodisiac

Honey, butter 15.5-

62.5

Svarnamaksika

[51,52]

Copper

pyrite

Anemia, inflammation, urinary

disorder, chronic fever, tuberculosis,

insomnia, epilepsy, scanty

mensuration, skin diseases, piles,

helminthiasis

Honey 125-

250

Haratala [24,53] Orpiment Diseases of abdomen, anemia, sinus,

epilepsy, disorders of blood, gout,

skin diseases, piles, tuberculosis,

leucoderma, blister, taeniasis,

eczema

Honey, butter 31-

125

7

powder. These procedures are repeated for particular number of cycles and the prepared

bhasma (ash) is collected [9,14,54].

1.4 Characteristic features of bhasma

The quality of bhasma is ascertained through various physical parameters like colour,

floatability, luster property, fineness, particle size and chemical test to check the

irreversibility of the bhasma to its original metallic form[12,54].

1.4.1 Physical parameters for assessment of quality of bhasma

1.4.1.1 Colour (Varna)

Each bhasma should possess a characteristic colour based on the metal chosen for the

preparation. Any difference in colour indicates that the bhasma has not been prepared

properly.

1.4.1.2 Floatability (Varitara)

To study the lightness and fineness of the bhasma, a pinch of the substance is sprinkled on

water and the floating tendency is observed. The floatability property depends on the

surface energy of the powder and the properly prepared bhasma floats on water.

1.4.1.3 Lusterless (Nishchandratvam)

Bhasma should not possess any luster while observed under sunlight. Luster indicates the

presence of free metallic form of the metal and indicates requirement for further

incineration before utilizing for therapy.

1.4.1.4 Fineness (Rekhapurnata)

To determine the fineness of the bhasma, a pinch of bhasma is taken and rubbed using

thumb and the index finger. A properly prepared bhasma fills the furrows of the finger tips.

8

1.4.1.5 Particle size

Bhasma should be in the form of a powder that resembles the pollen grains of Pandanus

odoratissimus flower.

1.4.2 Chemical parameters for assessment of quality of bhasma

The following tests are performed to determine the irreversibility of the bhasma.

1.4.2.1 Apunarbhavata

Bhasma is mixed with equal quantities of Abrus precatorius, honey, ghee, borax and

jaggery. The mixture is subjected to calcination as prescribed for the desired bhasma. The

presence of lustrous particles after calcination indicates improper preparation

1.4.2.2 Niruttha (Test for irreversibility)

Bhasma is mixed with a known quantity of silver and subjected to calcination. Increase in

weight of silver after calcination indicates improper preparation of the bhasma.

1.5 Controversies over metallic preparations

Even though bhasma are traditional Ayurvedic preparations there is a global perception that

these herbometallic preparations are not safe. This is attributed to the reports on the

presence of heavy metals in bhasma.

Saper et al. [55,56] detected lead, arsenic and mercury content in several US and Indian

manufactured Ayurvedic medicines. It was reported that several Indian manufactured

Rasashastra medicines could result in lead and mercury ingestions upto 100 to 10000 times

greater than acceptable limits. In addition, it was reported that one-fifth of both US-

manufactured and Indian-manufactured Ayurvedic medicines purchased online contained

detectable lead, mercury, or arsenic [55,56].

9

Kales et al. [57] compared the Ayurvedic lead poisoning with the common occupation-

based lead poisoning in terms of hematopoietic toxicity. Ayurvedic lead poisoning was

found to be more toxic than the occupation-based lead poisoning. It was strongly

recommended that users of Ayurvedic medicine must be screened for lead toxicity [57].

Dargan et al. [58] highlighted that several Ayurvedic medicines contain heavy metals that

are intentionally added during their preparation. The use of these medicines have been

reported to result in clinically significant heavy metal poisoning such as lead poisoning. It

was argued that the purification processes and the usage of herbs in the preparation would

not influence metal detoxification. In addition, concerns on improper labeling or

insufficient information on the labels of Ayurvedic medicines with respect to the

ingredients were also reported [58].

Different methods are adopted for preparation of the same bhasma. Hence, Kapoor et al.

[59] stated that there are no standard bhasma of the same metal. Calcination, being an

elaborate and time consuming process, bhasma cannot be prepared at short notice [60]. The

lack of clarity with respect to the chemical nature of the bhasma was also highlighted [59].

Surya et al. [31] suggested that the toxicity of bhasma would be influenced by the

preparation method, its chemical nature, vehicle used for administration, while reviewing

the safety and critical issues concerned with the use of Naga bhasma, a lead based

preparation. Lack of scientific validation of the preparation process, misinterpretation of

terms used in the literature were considered as probable reasons for improper preparation

of the bhasma. Toxic manifestations of Naga bhasma reported in the literature could be

attributed to deviations from the purification process and reduction in the number of

calcination cycles. The analysis of commercial Naga bhasma samples revealed batch-to-

10

batch and manufacturer-to-manufacturer variation indicating absence of widely acceptable

standard operating protocols [31].

Besides these toxicity issues pertaining to bhasma, the toxic effects of these metallic

preparations in case of improper preparations and the managements of the adverse effects

have been already portrayed in the literature and are tabulated below [14,60]

Table 1.3 Adverse effects of metals and the management of adverse effects.

There exists a correlation between the toxic effects of improper preparations described in

literature and several case studies reported with Ayurvedic drug poisoning. For instance,

Dargan et al. [58] reported high amount of lead in the blood of patients administered with

Metal Adverse effects Management of adverse effects

Mercury Skin disorders, osteoarthritis,

fainting, vomiting, diarrhea,

dyspnea

Administration of sulphur for 7

days

Gold Illness, weakness, impotency Chebulic myrobalans powder

should be administered for 3 days

Silver Anemia, itching, fever,

constipation, oligospermia,

weakness, headache, reduction in

potency

Sugar and honey should be

administered for 3 days

Copper Vomiting, fainting, hallucination,

skin disorders, burning sensation,

impotency

Dhanyaka fruit powder must be

administered with sugar candy

Iron Skin disorders, ureolithiasis,

spasmodic pain, burning sensation,

weakness

Embelia ribes (Vidanga) fruit

powder should be administered

with the juice of Sesbania

grandiflora leaves (Pragnakara)

Lead Diabetes, emaciation, anemia,

jaundice, skin disorders,

abdominal tumor, dyspepsia

Terminalia chebula powder with

sugar candy must be

administered for 3 days.

Tin Diabetes, skin disorders,

abdominal tumors, cardiac disease,

spasmodic pain, piles, cough,

weakness, vomiting

Gymnema sylvestre (fruit)

powder with sugar candy must be

administered

Zinc Diabetes, indigestion, vomiting,

hallucination

Terminalia chebula powder must

be administered with sugar candy

for 3 days.

11

Ayurvedic medicines. As an indication, these patients were confirmed with anemia. It is

evident from Table 1.3, that improper preparation of lead-based formulations result in

several adverse effects including anemia. It is imperative to understand the science

involved in the process of preparation prescribed in the literature, without which safety or

toxicity of herbo-metallic preparations cannot be generalized. The role of purifying agents,

herbs used in the preparation and process conditions must be investigated thoroughly

through appropriate, stage-wise physicochemical transformations.

1.6 Iron in Ayurvedic medicine

The usage of iron as medicine started as early as the vedic era. The usage of iron for internal

administration began in the 2nd century as evident from the use of Navayasa Lauha and

Lauha Rasayana [61]. A total of 293 formulations are based on iron oxide nanoparticles

(Lauha bhasma) and 85 formulations contain iron compounds such as iron pyrite, ferrous

sulphate and ochre [61]. Before the prevalence of Rasa sastra, iron was administerd for

internal use as Ayaskruti or Lauha Rasayana. The preparation involved heating thin plates

of iron to red hot condition and dipping in cow’s urine or amalaki juice. Later a unique

method involving purification and calcination steps was developed to convert iron to a

therapeutic form, called Lauha bhasma [61]. The use of iron as a supplement in modern

medicine began in 16th century. The existence of iron in blood was discovered only in 1713

[62].

1.7 Lauha bhasma

Lauha bhasma is one of the iron based herbo-metallic preparation prescribed in the

treatment of various diseases like anemia, jaundice, skin diseases, diarrhea, pain,

12

hyperacidity, diseases of abdomen, helminthiasis/worm infection, disorder of adipose

tissue, splenic disease, inflammation and asthma [9].

1.7.1 Lauha bhasma based formulations

Preparations containing Lauha bhasma as the major component are called Lauha kalpa.

They are prepared by mixing the Lauha bhasma with specific drugs and are processed to

powder form. In particular cases, wet grinding is also carried out [63]. Lauha kalpa are

classified into several types

Churna Lauha Kalpa : Dry grinding of the drugs with the Lauha bhasma and

converting into fine powder

Khalviya Lauha Kalpa : Wet grinding of herbal juice with Lauha bhasma

Lauha Rasakriya Kalpa : Boiling of Lauha bhasma with herbal juices or decoctions

until a paste consistency is obtained

Putapaka Lauha Kalpa : Lauha bhasma obtained after calcination

Some of the important Lauha Kalpa, their dose, vehicle and therapeutic uses are shown

in Table 1.4 [9].

Table 1.4 Important Lauha Kalpa, their dose, vehicle and therapeutic indications.

Lauha Kalpa Dosage

(mg)

Vehicle Disorders/diseases treated

Candanadi 250-500 Honey Intermittent fever, chronic fever

Dhatri 500-1000 Ghee, honey Gastric ulcer, anemia, jaundice,

hyperacidity, eye diseases, dyspepsia,

bleeding disorder

Pippalyadi 250 Honey Digestive impairment, emesis, hiccup,

asthma

Putapakva

visama

jvarntaka

250 Pippali,

honey

Fever, malabsorption syndrome,

splenic disease, jaundice, anemia,

inflammation, intermittent fever,

asthma, diarrhoea

13

Pradarantaka 500 Sugar,

honey, ghee

Digestive impairment, gastric ulcer,

anemia, pelvic pain, lower backache,

pain in female genital tract,

tastelessness, cough, asthma,

weakness

Pradarari 1000-

2000

Kusamula

kasaya, milk

Excessive vaginal discharge, pain,

pelvic pain, digestive impairment

Yakrdari 250-500 Pippali

curna, honey

Diseases of abdomen, abdominal

lump, jaundice, diseases of liver,

splenic disease, chronic fever, cough,

asthma, dyspepsia, weakness

Rohitaka 250 Honey Splenic disease, liver disease,

inflammation

Vidanga 500 Honey Digestive impairment, tastelessness,

pain, piles, inflammation, fever,

asthma,

Vidangadi 250 Honey,

buttermilk

Jaundice, anemia, inflammation

Saptamrta 250 Honey,

ghee, milk

Emesis, pain, fever, obstruction in

urinary tract, cataract, inflammation

Sarvajvarahara 250 Honey Chronic fever, splenic disease, liver

disease, ventricular hypertrophy

The prior-art on the preparation of Lauha bhasma, its toxicity and pharmacological

activities are discussed in the subsequent paragraphs.

1.8 Review on methods of preparation

A systematic review on the preparation of Lauha bhasma was carried out by Bhanuprakash

[4]. Lauha bhasma obtained after 1000 calcination cycles was suggested to be more potent,

consisting of almost pure iron particles arising due to the reduction of the oxides by carbon

present in triphala extract [4].

Pandit et al. [64] prepared Lauha bhasma as per the procedure described by Nagarjuna, in

which 545 calcination cycles was suggested. The preparation involved initial calcination

of iron in sarava samputa for 12 hours using cow dung cakes, followed by immersion in

cow’s urine for three months for purification. The cow’s urine was discarded and the iron

14

dried under sunlight. The purified iron was boiled with triphala kasaya and again immersed

in cow’s urine for 21 days. The iron was then ground with triphala kasaya and subjected

to calcination. The role of cow’s urine, triphala kasaya and calcination cycles in the

preparation of Lauha bhasma was not investigated [64]. While the elemental composition

of Lauha bhasma obtained after 545 calcination cycles was reported, the crystalline phase

of raw material and that of Lauha bhasma were not been reported. In case of elemental

composition, the percentage of iron was found to decrease with the number of calcinations

cycle. Besides iron, elements such as Mn, Cu, Zn & Co were also found at levels lower

than 1000 ppm. However, the higher concentration of lead (5600 ppm) in the Lauha

bhasma obtained after 545 calcination cycles raises question about its safety. The choice

of plants used during the calcination cycles is driven by the disease to be treated [64], as

shown in Table 1.5

Table 1.5 Plants used in the preparation of Lauha bhasma for specific diseases.

S.no Plant Botanical name Disease

1 Juice of Bidari Ipomoea digitata Impotency

2 Lime juice Citrus bergamia Loss of appetite

3 Decoction of Sirisha Albizzia lebbeck Loss of complexion

4 Juice of Bala Sida cordifolia Rheumatism

5 Juice of Bharangi Clerodendron

siphonanthus

Bronchial asthma

6 Decoction of

Rohitaka

Amoora rohituka Splenomegally

7 Juice of

Ashwagandha and

Jatamansi

Withania somnifera,

Nardostachys

jatamansi

For the wasting

disease

Sarkar et al. [65] prepared Lauha bhasma by employing electrical muffle furnace for

calcination. The quality of the bhasma was checked by employing classical physico-

chemical parameters. Though FeS was reported to have been formed, neither X-ray

15

diffraction data nor elemental composition data was acquired to confirm the same [65].

The elemental analysis must have been carried out as mercury was also used during the

preparation.

Rajendra Prasad et al. [66] performed pharmaceutical and analytical investigations on

Lauha bhasma prepared through seven calcination cycles using cow dung cakes as heat

source. While the raw material contained 96.82% of iron, the final product contained

89.48% of iron only, implying that other elements have been incorporated during the

preparation process. However, no efforts were made to identify the chemical nature of

Lauha bhasma through X-ray diffraction spectra. Even though the desired property of the

bhasma was confirmed by employing Ayurvedic parameters such as apunarbhavata

(incapable of regaining original metallic form) and nirutta (inability to regain metallic

form), the science behind the purification and calcination process has not been explored

[66].

Neetu Singh et al. [67] carried out experiments to determine the appropriate calcination

temperature for the preparation of Lauha bhasma in an electrical muffle furnace. The

preparation commenced with the samanya sodhana, followed by vishesha sodhana,

bhanupaka and sthalipaka as per classical texts. The intermediate after sthalipaka was

divided into two batches. Calcination was carried out at 800°C for the first batch, while a

calcination temperature of 600°C was used for the second batch. The sample prepared by

calcination at 800 °C turned hard and regained the metallic property after 5-6 calcination

cycles. The sample prepared after 20 cycles of calcination at 600°C floated when sprinkled

on water, thereby possessing one of the characteristic features of bhasma prescribed in

16

Ayurvedic texts. However the termination of calcination cycle was not justified through

analysis of elemental composition or diffraction spectra [67].

Virupaksha et al. [68] carried out experiments on the preparation of Lauha bhasma in an

attempt to validate the steps involved. Samanya sodhana, vishesha sodhana, bhanupaka

and sthalipaka were performed according to classical text, while 20 cycles of calcination

was performed in an electrical muffle furnace at 650°C rather than using cow dung cakes.

Muffle furnace was used for calcination to maintain uniform temperature across all

calcination cycles, rendering calcination independent of vagaries of monsoon and seasonal

variations. Though a hypothesis was proposed for physico-chemical changes during

calcination, the same was not validated using experiments[68].

A compilation of raw materials and procedures adopted by various researchers is provided

in Table 1.6.

Table 1.6 Procedures followed by various researchers in the preparation of Lauha

bhasma.

Investigators Raw material

Sarkar et al. [65] Scraps of wrought iron

Rajendra Prasad et al. [66] Loha

Neetu singh et al. [67] Lauha

Viruprakasha et al. [68] Tikshna Lauha (Iron scrap)

17

Investigators Bhanupaka (Exposure to sunlight)

Sarkar et al. [65] Not performed

Rajendra Prasad et al. [66] Purified Lauha mixed with triphala kasaya and exposed

to sunlight till the fluid evaporates.

Process repeated for 3 times.

Neetu singh et al. [67] Purified Lauha mixed with triphala kasaya and exposed

to sunlight till the fluid evaporates.

Process repeated for 7 times.

Investigators Samanya sodhana & Vishesha Sodhana

( General & Special Purification)

Sarkar et al. [65]

Rajendra Prasad et al. [66]

Neetu singh et al. [67]

Viruprakasha et al. [68]

Heated to red hot and quenched in sesame oil, butter

milk, cow’s urine, sour gruel, kulatha decoction &

triphala decoction.

Each process repeated for 7 times.

Temperature not recorded.

Investigators Sthalipaka (Heating in an iron pan)

Sarkar et al. [65] Not performed

Rajendra Prasad et al. [66] Lauha mixed with decoction prepared using Butea

monosperma, Ricinus communis and Eclipta prostrata.

Heated in sthali (iron pan) until the decoction evaporates.

Process repeated for 3 times.

Neetu singh et al. [67] Lauha mixed with triphala decoction

Heated in sthali (iron pan) until decoction evaporates.

Process repeated for 7 times.

Viruprakasha et al. [68] Lauha mixed with triphala decoction

Heated in sthali (iron pan) until decoction evaporates.

Process repeated for 7 times.

18

Investigators Putapaka (Calcination)

Sarkar et al. [65] Purified iron mixed with 1/12th cinnabar, levigated with

aloe gel for 6 hours.

Pellets prepared, dried and sealed in sarava samputa.

Calcination process performed in an electrical muffle

furnace for 3 hours.

Temperature not mentioned.

Total number of calcination-7

Rajendra Prasad et al. [66] Cinnabar powdered well and 12 parts of Lauha added,

mixed well

Grounded with aloe gel, cakrikas were prepared, dried

and sealed.

Calcination process using cowdung cakes. Total

number of cow dung cakes used-1000.

Temperature recorded.

Total number of calcination -7

Neetu singh et al. [67] Purified iron grounded with triphala kwatha

Calcination process performed in an electrical muffle

furnace for 1 hour at two different temperatures 600ºC

for one batch and 800 ºC for another

20 calcination for Batch I and 22 calcination for Batch

II

Viruprakasha et al. [68] Purified iron grounded with triphala kwatha

Calcination process performed in an electrical muffle

furnace for 1 hour at 650ºC

Total number of calcination -20

Neetu Singh et al. [69] employed powder X-Ray Diffraction and vibrating sample

magnetometry as tools for characterization of Lauha bhasma. Sample obtained after 20th

calcination cycle was found to be more crystalline than that obtained after sthalipaka. The

final product obtained after 20 calcination cycles was found to contain both -Fe2O3 and

Fe3O4 in equal proportions. The magnetic nature of samples was confirmed through

vibrating sample magnetometry [69].

Neetu Singh et al. [70] carried out measurement of particle size and elemental composition

of Lauha bhasma. The incorporation of trace elements in the finally prepared bhasma was

19

confirmed through atomic absorption spectroscopy (AAS) and energy dispersive analysis

of X-rays (EDAX). Intermediates obtained after bhanupaka and sthalipaka contained

appreciable amount of carbon due to the addition of triphala kasaya. Potassium was found

to be incorporated during calcination. While the reduction in iron content was attributed

to its oxidation, the reduction in particle size was attributed to grinding and calcination

[70].

Bhargav et al. [71] had reported preparation of Lauha bhasma using two different raw

materials: magnetite iron and iron turnings. Bhasma prepared using magnetite iron was

named 'Kanta Lauha' (two batches), while that prepared using iron turnings was named

'Teekshana Lauha' (one batch). The number of calcination cycles employed for preparation

of 'Kanta Lauha' and 'Teekshana Lauha' were 18 and 20 respectively. Though the authors

interpreted powder X-ray diffraction patterns of 'Kanta Lauha' and 'Teekshana Lauha' to

be those of -Fe2O3 , '2θ' value for the major peak (~ 36° ) matched well with those of-

Fe2O3 or Fe3O4. There was significant batch-to-batch variation in iron content in the two

batches of Kanta Lauha prepared. The iron content in one batch of Kanta Lauha increased

during preparation, while that in the other batch decreased [71].

1.9 Review on Toxicity and Therapeutic efficacy of Lauha bhasma:

Sarkar et al. [72] performed investigations on toxicity of Lauha bhasma in experimental

animals followed by recovery studies. Lauha bhasma was administered to experimental

animals (Albino rats) at a dosage of five times the therapeutic effective dose. The drug

was administered for 60 days and 45-days recovery period was observed. Hyperglycemia

was observed in Lauha bhasma treated group. The increase in sugar level might be

attributed to liver damage in Lauha bhasma treated group as evidenced from

20

histopathological sections of liver that confirmed mild to moderate fatty degenerative

changes, necrosis, central vein congestion and sinusoidal dilation. However, glucose level

was restored to normalcy during the recovery period. In addition, normal cyto architecture

was also observed. During the recovery period, hematological parameters of Lauha bhasma

treated animals were found to be normal with respect to control group. However decrease

in hemoglobin was observed which could be attributed to pseudo iron deficiency caused

by abrupt stopping of the Lauha bhasma administration [72].

Pandit et al. [64] studied the haematinic activity of Lauha bhasma (obtained after 50

calcination cycles) in Phlembotomy-induced iron deficiency anemia in test animals. Lauha

bhasma treated animals showed significant recovery than the control and ferrous sulphate-

treated groups. Apart from eradicating the iron deficiency, multi-elemental combination of

Lauha bhasma fulfilled various elemental demands of the animal also. Hemoglobin level

was increased in Lauha bhasma-treated animals. The efficacy of Lauha bhasma was

comparable to that of ferrous sulphate as evidenced from haemotological and biochemical

parameters [64].

Sarkar et al. [73] evaluated the haematinic potential of Lauha bhasma against HgCl2-

induced anemia in test animals at a therapeutic dose of 11mg/kg body weight for 30 days.

Prior to administration of drugs, hematological parameters including RBC, hemoglobin

and hematocrit showed significant decrease in mercuric chloride treated group when

compared to control group. Bhasma administration resulted in restoration of these

parameters highlighting the hematinic potential of Lauha bhasma [73].

It is pertinent to note that the therapeutic studies reported above did not elucidate the

molecular mechanism by which the Lauha bhasma restored the hematological parameters.

21

The antibacterial activity of Lauha bhasma was investigated using disc diffusion method.

For this purpose, Lauha bhasma was dispersed in water, methanol and acetone separately.

Lauha bhasma was found to exhibit strong anti-bacterial activity against S.typhi,

S,typhimurium, S.aureus, P.vulgaris [74].

In vivo genotoxicity of four Lauha bhasmas was assessed through study of chromosomal

damage and DNA strand breaks. Micronucleus and comet assay was used for this purpose.

A single dose of 2000mg/kg of Lauha bhasma was administered to Wistar rats and

sacrificed after 24 hours. Cyclophosphamide was used as the positive control.

Micronucleus assay confirmed that there was no chromosomal damage in Lauha bhasma

administered groups. There was no significant difference in Poly chromatic erythrocytes

to normo chromatic erythrocytes ratio between control and Lauha bhasma treated groups.

Comet assay revealed that the DNA damage percentage were similar for Lauha bhasma

treated and control groups [75].

1.10 Toxicity of iron

Iron, the fourth abundant element of earth’s crust is well known from ancient times. Even

though it plays an essential role in biological systems, its toxic effect results in male

sterility, skin disorders, cardiac diseases, calculi, colicky pain, nausea, death and

tumorogenesis. Under normal circumstances, Fe2+ acts as an electron donor and Fe3+ acts

as an electron acceptor in many biochemical reactions. But this redox capability of iron is

also a potential biohazard as the same can catalyze the formation of free radicals. Iron

toxicity is mainly based upon Fenton and Haber Weiss chemistry. The iron-catalyzed

Haber–Weiss reaction, which makes use of Fenton chemistry, is considered to be the major

mechanism by which the highly reactive hydroxyl radical is generated in biological system.

22

Reactive oxygen intermediates are inevitable byproducts of aerobic respiration and emerge

due to incomplete reduction of dioxygen in mitochondria. Free radicals can easily interrupt

with lipids, proteins and nucleic acid metabolism resulting in oxidation of proteins,

peroxidation of membrane lipids and modification of nucleic acids[76–78]. Direct

interference of reactive oxygen intermediates may alter the gene activation or inactivation

process, which results in the structure and functional modification of a particular protein.

ROI may inhibit the anti-oncogenes and induce oncogenes that lead to changes in

apoptosis leading to cancer [79–81].

1.10.1 Toxic Manifestations of Iron:

Various reports associated with iron toxicity are tabulated below (Table 1.7)

Table 1.7 Toxic manifestations of iron

Authors Form of Iron Organs

damaged

Pathological findings

Elseweidy et al.

[82]

Ferrous

sulphate

Brain Lipid peroxidation. Meningeal

hemorrhage, congestion and edema due

to excessive iron overload.

Sobotka et al. [83] Iron rich diets Brain Dose dependent toxicity and behavioral

changes.

Maaroufi et al. [84] Ferrous

sulphate

Brain Behavioral impairments combined with

iron accumulation

Velez-Pardo et al.

[85]

Ferrocene PC-12 cells Apoptosis in PC-12 cells

Erichsen et al. [86] Ferrous

fumarate

Intestine Excess iron deposition with increased

colitis score

Carrier et al. [87] Ferrous

fumarate

Intestine Heavier rectal bleeding and shortening

of the colon

Lund et al. [88] Iron rich diets Intestine Free radical generation in colon and

lipid peroxidation in caecum

Ozguner et al. [89] Iron-sorbitol Liver Hepatocytes were seen with iron

overload by characteristic yellowish

brown deposition

Al-shaikh [90] Ferrous

fumarate

Liver &

Kidney

Disruption of liver and kidney cyto

architecture

23

Machado et al. [91] Iron peptide

complex &

Iron sulphate

Liver &

spleen

Excess iron deposition

Horne et al. [92] Carbonyl iron

diet

Pancreas Chronic pancreatitis

1.11 Iron metabolism:

In general, iron is distributed in animal and plant tissues. Iron is present in the form of

heme, ferritin and hemosiderin in animal tissues. In case of animal derived foods such as

milk and egg, iron is associated with specific proteins such as lactoferrin (milk),

ovotransferrin (egg white) and phosphovitin (egg yolk). In plants, iron is distributed in the

form of metalloproteins, plant ferritin and complexed with structural or storage

components [93]. The bioavailability of non-heme iron is influenced by various factors in

the intestinal layer, whereas the heme iron gets absorbed directly as metallo-protein

porphyrin complex. Gastric hydrochloric acid plays a significant role in the solubility of

iron in non-heme food. Iron absorption is enhanced by organic acids such as ascorbic acid,

malic acid, lactic acid and proteins derived from animal sources. Ascorbic acid reduces the

insoluble Fe3+ to more soluble Fe2+ and maintains the acidic nature of the gastro-intestinal

tract. The potent inhibitors of iron absorption are dietary fibers, phytic acid, inorganic

phosphates, tannins and polyphenols [93,94].

Besides, iron is an important element in our biological system. Iron plays a major role in

electron transport and is an important constituent of several metallo proteins such as

hemoglobin and myoglobin. About 3-5 g of iron is present in the human body of which a

major part is utilized by RBCs for the synthesis of heme. Iron is also located in enzymes

like oxygenases and peroxidases [95–97].

24

Hemoglobin in erythrocytes constitutes the largest iron pool. Hemoglobin plays a

significant role in transporting oxygen from lungs to the peripheral tissues and carbon

dioxide from tissues to the lungs [98]. Normally 25 mg of iron is required for

erythropoiesis. Hepatocytes and Macrophages constitute the second largest iron pool that

contain storage iron. Myoglobin, the third largest iron pool contains heme moieties that are

necessary for normal muscle and cardiac function [97]. Specific transporters and

mechanisms exist through which iron is utilized in the biological systems by retaining it in

soluble, non-toxic form [99].

1.11.1 Iron transport across the intestine

The absorption of iron begins in the enterocytes of small intestine. Dcyt-b (Ferric

reductase) present in the brush borders of intestinal villi reduces the ferric complexes (Fe3+)

to ferrous form (Fe2+). The reduced Fe2+ is transported across the brush border membrane

by the proton-coupled divalent metal transporter 1 (DMT-1) and enters an unknown

compartment in the cytosol. Fe2+ is either stored as Ferritin or transported across the

basolateral membrane by ferroportin where the membrane bound copper oxidase

Hephaestin (Hp) promotes Fe2+ to Fe3+conversion. Fe3+binds to circulating transferrin in

the blood [97-104]. Ferritin (a storage protein) and transferrin (a glyco protein) play

significant roles in cellular iron metabolism. Each ferritin molecule is composed of 24

polypeptide subunits which form a basket like structure that can hold up to several thousand

atoms of iron [105,106].

1.11.2 Cellular Iron metabolism

Transferrin enables the transport of iron to the site of absorption, storage and utilization.

Transferrin is an 80 kDa glycoprotein present in plasma that contains two specific, high

25

affinity binding sites for Fe3+ at neutral pH. However the affinity decreases when the pH

decreases. Moreover Fe2+ does not bind specifically to these sites [98,104,107].

Iron released from enterocytes, hepatocytes and macrophages are assimilated by transferrin

for delivery to cells. Iron-bound transferrin binds to the specific transferrin receptor TfR1

on the cell surface [108-111]. By endocytosis process, it is internalized leading to the

formation of endosomes.

Acidification of endosomes at pH 5.5 releases iron from the transferrin. Iron is then

transported through DMT 1 across the endosomal membrane. Iron is reduced from Fe3+to

Fe2+ state in cytosol, catalyzed by Steap 3, a protein of Steap family[98]. Thus it is either

used for hematopoiesis process or the excess iron is stored in ferritin [97-104].

Figure 1.1 Schematic representation of normal iron metabolism.

26

1.11.3 Regulation of cellular iron metabolism

The uptake of transferrin into cells is exquisitely controlled by intracellular iron pools that

regulate proteins involved in iron metabolism at the post transcriptional level. The Iron

responsive elements (IRE-IRP system) play major roles in regulating cellular iron

metabolism. Iron responsive elements (IRE) are the stem loop structures present in the 3´

end of transferrin receptor m-RNA and IRP are the iron regulatory proteins. When the

cellular iron content is low, the IRP binds to the IRE and prevents the degradation of TfR1

RNA, thus promoting the translation process of TfR1. As a result more TfR1 is produced,

which results in uptake of iron by transferrin inside the cell. When the intracellular iron

concentration increases sufficiently, IRP does bind to the IRE region of TfR1 m-RNA and

inhibit the TfR1 translation process[97-101,112,113].

1.12 CONCLUSION

Though the Ayurvedic system is practiced for several centuries, the science involved in the

process of preparation of metallic drugs and their pharmacological actions are not

completely revealed. Several factors such as raw material, purifying agents used, herbs

used, method of preparation, number of calcination cycles, temperature influence the

physico-chemical characteristics of bhasma, which may determine its toxic and therapeutic

effect. Hence it is imperative to understand the process thoroughly and scientifically

ascertain the role of the factors that influence the preparation. Improper preparation of

Lauha bhasma through adoption of abridged procedures and lesser number of calcination

cycles may probably lead to toxic manifestations of the iron and probably result in various

pathogenic conditions. Though the mechanism of iron metabolism is well-understood, the

molecular targets of the therapeutic action of Lauha bhasma needs to be identified.

27

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