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9Chemometric analysis: A novel tool for herbal drug analysis and designing 209
there was no splitting of the other cluster even at lower level. The cluster was of PG-4, PG-5 and PG-6, which showed very similar guggulsterone content. The content of the other five samples was different from this group, where samples PG-2, PG-3 forming one cluster were different from first level splitted cluster of RG-D, PG-7 and PG-1. Clus­tering of different purified samples on the basis of guggulsterones are illusterated well by the dendrogram in Fig.9.5.
Table 9.5: Cluster membership.
S.No. Case Clusters
. RG-D . PG-  . PG-  . PG-  . PG-  . PG-  . PG-  . PG-
Case 0 5 10 15 20 25 Label
RGD PGG PGA PGB PGC PGE PGF PGD
Num
1 8 2 3 4 6 7
5
Fig. 9.5: Dendrogram showing clustering of different purified samples.
9.4.5 Principal component analysis (PCA) for similarities between differently purified samples
The purified guggulu samples through different methods show differenc
es among themselves to varying extents as inferred from HPLC analysis. The present analysis to check similarity was based on the assumption that each peak in the HPLC chro­matogram represent one component or group of components (unresolved under test condition) and peaks at same retention times in different chromatograms of different purified samples represent the same component(s). In order to evaluate the discrimi­nation ability of the different constituents, PCA was employed using the peak areas of all peaks as input data. On the basis of eigen values, the first two principal com-
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ponents PC1 and PC2 were used to provide a convenient visual aid for representing gross inhomogeneity in the data sets. It was clear from the variance matrix shown in Table9.6 that the first two principal components contributed 97.88% of the variance, which was further confirmed from the scree plot as the elbow of the plot lay in the first two component regions.
Table 9.6: Total variance matrix in PCA analysis of differently purified guggulu samples.
Component Initial eigen values Extraction sums of squared loadings
Total Percent
variance
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cumulative percentage
Total Percent
variance
Cumulative percentage
The samples were grouped in two categories based on their values in rotated com­ponent matrix scores (Table 9.7). The samples which had significant scores for the component were extracted in the respective component (Table 9.8).
These results were in agreement with the cluster analysis. The results of HCA and PCA producing the same inference validated these results and provided proof to the results of analysis of these samples in similarity assessment.
The score plot was made using the first two principal components and is shown in Fig.9.6.
Table 9.7: Rotated component matrix in PCA analysis of differently purified guggulu samples.
Sample Component

RG-D
PG- . . PG- . . PG- . . PG- . . PG- . . PG- . . PG- . .
. .
9Chemometric analysis: A novel tool for herbal drug analysis and designing 211
Table 9.8: Groupings of samples on the basis of their component scores.
Components extracted

PG- RG-D PG- PG- PG- PG-
10
8
6
4
Eigenvalue
2
PG- PG-
Scree plot
0
1234
Component number
Fig. 9.6: Scree plot between eigen values and components in PCA analysis of differently purified
guggulu samples.
5678
Figure 9.7 is a graphic display of similarities in the components eluted at the respec­tive retention times; taking into consideration the AUC of different peaks observed in HPLC analysis of differently purified guggulu samples and making comparison. Each dot in the figure represents retention times of different peaks. These dots are expected to cluster at one point, had there been no difference in chromatograms of all samples with respect to presence / absence of peak and also in the values of their AUCs. Simi­larly, if all peaks had been different in different samples, their dispersion would have been wide apart on different planes. The analysis of Fig.9.7 clearly indicates that the peaks at retention times 1.92, 11.11 and 13.30 are outside the cluster showing dissimi­larity, whereas most other peaks appeared in almost close cluster indicating similar­ity with respect to most other peaks. The three peaks appearing at a distance from the cluster indicates that these are the points of maximum variation in purified guggulu samples. It is interesting to note here that the peaks at retention time 11.11 and 13.30, which are outside the cluster showing dissimilarity, are the peaks of E- and Z- gug-
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3.00000
2.00000
1.00000
*
Principle component 2
.00000
–1.00000
–2.00000–1.00000 1.00000
Principle component 1
Fig. 9.7: Relation of different samples based on principal component score using retention times.
1.92
2.3
3.07
4.18
RT
7.96
13.3
8.76
14.46
9.9
19.15
11.11
*
2.00000 3.00000.00000
gulsterone, respectively. This clearly shows that the purification technique actually varies the content, as validated by the chemometric analysis.
9.5 Conclusion
The results of chemometric analysis clearly depict that the various guggulu samples from different geographical regions vary considerably with few showing resemblance to one another. Accordingly, we can target procurement of this particular drug of high repute from various regions. This study has led to the following conclusions:
1. The content of guggulsterones varies in different samples based on the geograph-
ical source.
2. The genuine cultivated samples from Ajmer, that is RG-A
resembled RG-J, the sample from Jalandhar which can be easily replaced with the
genuine drug.
3. The content of guggulsterones alters significantly with the type of purification
process employed.
4. PG-4, PG-5 and PG-6 have the maximum content of guggulsterones and thus are
the best methods of purification for this drug out of the seven methods mentioned
in Ayurvedic texts.
and RG-A2, closely
1
9Chemometric analysis: A novel tool for herbal drug analysis and designing 213
It has been mentioned in Ayurvedic texts that administration of raw guggulu may sometimes lead to skin rashes, irregular menstruation, diarrhoea, headache, mild nausea, and with very high doses, liver toxicity [8]. In order to overcome the side effects of raw guggulu, Ayurveda describes a number of purification processes (shod­hanvidhi) in different ‘dravyas’ i.e., fluids, which not only takes care of the adverse effects but also enhances the therapeutic activity. It is also mentioned in Ayurvedic texts that guggulu must be purified before incorporation into herbal formulations. There are a large number of commercial polyherbal anti-inflammatory formulations which are using guggulu as the chief ingredient. Thus, we can effectively design the new polyherbal guggulu formulations based on the conclusions drawn out of this study. The geographical source and the method of purification of the drug can be cau­tiously chosen to enhance the pharmacological activity of guggulu. The chemometric methods used in the study can be effectively employed in designing a multifaceted polyherbal formulation which will suffice to cure variety of inflammatory disorders.
References
[1] Kong WJ, Zhao YL, Xiao XH,et al. 2009. Quantitative and chemical fingerprint analysis for
quality control ofRhizoma Coptidischinensisbased on UPLC-PAD combined with chemometrics methods, Phytomedicine 16, 950–959.
[2] Du Jing WD, Huang L, Chen S, Qin M. 2011. Application of chemometrics in quality evaluation of
medicinal plants, Journal of Medicinal Plants Research, 4001–4008.
[3] Gupta V, Singh R, Singh G, Singh R, Singh H. 2011. An introduction to principal component
analysis and its importance in biomedical signal processing, International Conference on Life Science and Technology,3, 31–33.
[4] Sangle VD, Nadkarni SD, Vahalia MK, Darp MS. 2004. The study of effect of ayurvedic
processing of Commiphora wightii on gastric irritancy index in experimental animals, Indian Drugs 41, 268–271.
[5] Anonymous. 2003. The Ayurvedic Formulary of India. Edn 2, Vol. I, Department of Indian
Systems of Medicine and Homeopathy, Ministry of Health and Family Welfare, Government of India, New Delhi, pp. 63–71.
[6] Karan M, Sarup P, Suneja V, Vasisht K. 2012. Effect of traditional Ayurvedic purification
processes (sodhanvidhi) of guggulu on carrageenan-induced paw oedema in rats, Journal of Pharmaceutical and Biomedical Sciences 21, 5.
[7] Karan M, Sarup P, Vasisht K. 2013. Evaluation of antioxidant and antinociceptive potential of
raw and purified guggulu, Journal of Pharmaceutical and Biomedical Sciences 31, 1150–1158.
[8] Masten SA. 2005. Gum guggul and some of its steroidal constituents: review of toxicological
literature. Integrated Laboratory Systems, Inc. Research Triangle Park, North Carolina, USA. Document prepared for National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health, U.S Department of Health and Human Services, February 2005; 2, 1–49.
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Subash Chandra Sahoo, Ramesh Kataria* and S.K. Mehta
10 Copper and its complexes: A pharmaceutical
perspective
Abstract: Copper came into view mainly due to its enzymatic function in the human
body and directly or indirectly plays a major role in regulating human activities like maintenance of the immune system, cancer, osteoporosis, arthritis, skin disease and heart problems etc. All the disease mentioned are also governed by copper and depend upon the amount of copper consumed in daily life. Therefore, knowing about the optimum intake of copper in the diet is very crucial for human health. Copper and its complexes also possess a good recognition in the field of medical science as bioactive agents. These copper dependent bioactive agents, due to their activity, are attracting interest from scientists for their use as prospective drugs for the treatment of several diseases. In this chapter we have tried to summarize the role of copper and its complexes with reference to toxicity level, sources, progress in the development of drugs and various important discussions about the mode of action.
10.1 Introduction
Copper is an important transition metal element with atomic number 29 in the periodic table that is universally engaged in biological systems via diverse activi­ties including embryonic development, mitochondrial respiration and regulation of hemoglobin levels etc. [1]. It pageants substantial biochemical action as a trace element or as a constituent of various exogenous compounds (copper complexes of anthranilic acid, 3,5-diisopropylsalicyclic acid, aspirin and carboxylic acid etc.) in humans. It plays a major role as a cofactor for abundant enzymes, such as cyto­chrome coxidase, tyrosinase, ceruloplasmin, albumin, including many biomole­cules and nucleic acids [2, 3]. In earlier times, copper was used as a sterilizing agent for drinking water, burns and wounds, headaches, and itching. Hippocrates, the father of modern medicine, described copper as a curing agent for leg ulcers [4, 5]. Celsus et al. described the use of copper and various copper composites for the han­dling of venereal disorders. The first disclosure about the role of copper and copper compounds in the immune system was confirmed when workers in copper mining had strong immune resistance to cholera during cholera epidemics that broke out in Paris [4, 5]. The French physician, Luton, reported that salt of copper (copper acetate), functions well in taking care of arthritis patients both by external and inter­nal use of copper acetate. The pharmacological actions of copper compounds were noticed in 1895 to treat various diseases including chronic diarrhea, dysentery and cholera. A researcher from Germany also mentioned in his findings that workers in copper mining were not touched by arthritis until they ended their employment in
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mining and same observation was favored by the French physician Luton’s finding in 1885 [4, 5]. W. B. Saunders Company in their report recommended the dose of 0.5g/ glass of water of copper sulfate to induce vomiting [4, 5]. It was also established that copper complexes deliberately boost healing time of ulcers and wounds. In addi­tion, it was shown that the number of radioisotopes of copper, play a very important role in radiotherapy and imaging applications [6]. Researchers have devoted more attention to exploring the chemistry of copper and its complexes, owing to its poten­tial and attractiveness for drug development in medical sciences. However, most of the inorganic salts of copper are toxic and can form a wide range of compounds with variable oxidation states including (I), (II), and (III). Among these, (II) is the most stable state for biological activity while the others (I and III) are less stable in while forming the complexes of 4, 5, and 6 coordinated species [7]. Again, the mode of action of copper is totally different from its organometallic counterpart as a drug, because copper/copper ions alone cannot act as a drug, but when combined with some organic moiety then response is more effective. New emerging research areas like nanoscience and nanotechnology are adding new perspectives to utilizing copper and its complexes in drug delivery systems [8, 9]. In earlier times, our ances­tors discovered the importance of copper through experience and keen perception. Through substantial progress in biological science, the importance of copper in rela­tion to human health has been fairly improved to date, however, many issue are still unclear and need to be explored. Researchers are still in action to explore the func­tion of copper in human health so as to exploit all possibilities of copper compounds as drug for human betterment. This chapter elucidates the current states of advance­ment of copper-based composite materials for medical applications.
10.2 Source of dietetic copper
There are several sources of copper in the human diet, but plants play a leading role toward attaining human beings’ necessary copper supply. The following plants are the chief accumulators of copper from soil and water: Aeolanthus biformifolius,
Athyrium yokoscense, Azolla filiculoides, Callisneria americana, Eichhornia crassipes, Haumaniustrum robertii and Bacopa monnieri. Therefore, the main sources of copper
for the human diet are grains, sesame seeds, cashew nuts, soybeans, mushrooms, sunflower seeds, lentils, beans, nuts, potatoes, green leaves, meat, dried fruits, black pepper, yeast and oysters. Human breast milk has the maximum concentration of copper (0.25 to 6.0mg/l) [10]. Among the manmade foods, the main sources of copper are cocoa, legumes, liver, wine (red) and organ meats [11]. However, dietary and life­style factors can play a very crucial role in maintaining the copper balance by pre­venting to acquire adequate copper either by escalated excretion or reduced absorp­tion. People who always prefer to drink soda or bottled water instead of spring water or natural well water, are keeping themselves away from a good supply of dietetic
10Copper and its complexes: A pharmaceutical perspective 217
copper. Generally, people who reside at high altitude in mountain valleys above sea level have the benefit of copper enriched water from natural glaciers.
10.3 Recommended concentration of copper for human diet
We know that copper is a crucial element which imparts a key role in human health; to establish a recommended limit, it is mandatory to know the concentration of copper in terms of surplus and deficiency in biological activities. In the continuation of ongoing attempts to fix the copper quantity in human diet, the Food and Nutrition Board (FNB) recommended 1.5 to 3.0mg copper for adults per day [12]. The board also suggested that the present data on copper is enough to calculate the range of requirement, but not sufficient to establish a Recommended Daily Allowances (RDA) [12, 13]. An amount of copper more than the 1.5 to 3.0mg daily intake in diet is toxic and responsible for the malfunctioning of the human system [12, 13]. The delay in notifying the significance of copper was finally pointed out by Klevay and Medeiros in 1996 [14]. They re-evaluated the previous ten versions of the recommended per day guidelines and found that the recommended amount of copper per day in 1943 was 1–2mg, which was significantly different from the 1958 recommendation of 2mg per day for adults. On the other hand, in 1989 [11] recommended intake of copper was again found to be 1.2mg per day for male adults and 0.9mg/day for females, which was quite low compared to the recommended 2mg per day. The intake value kept on changing from time to time by different agencies like 2mg/day by U.S. Food and Drug Administration, 1.5–3mg by Natural Research Council (1989), followed by the WHO suggesting 10mg/day consumption as the reasonable higher side limit of copper [12]. Suddenly, a decrease in an intake value to 0.9mg/day became a central discussion due to a much lower recommended value [11]. But in 2010, Chambers et al.’s [15] find­ings recommended an optimum consumption of copper to be 2.6mg/day to avoid copper deficiency. The recommended low intake of copper was not an acceptable value as the estimated routine loss of copper is about 1.3mg per day [11]. In active males, there is additional 0.34mg/day loss of copper due to sweating, shown by Jacob in 1981 [16]. Keeping in view all the facts, Hoogenraad [17] reported that a normal person’s intake of a higher quantity of copper has an unimportant or no effect on its absorption because under normal conditions, the amount of copper absorbed in the upper gastrointestinal tract is generally ∼0.5mg per day and the remaining amount of copper from dietary intake is directly eliminated without entering pathways of absorp­tion. The Food and Nutrition Board of the National Academy of Sciences recommends daily copper amounts according to age, 0–6months: 0.2mg; 6–12months: 0.22mg; 1–3years: 0.34mg; 4–8years: 0.4mg; 9–13years: 0.7mg; 14–18years: 0.89mg; for adults: 0.9mg; pregnant women: 1.0mg and breast feeding women: 1.3mg [18]. The Board also recommends around 10mg for adult men and women per day as the higher
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consumption limit [18, 19]. Study also reflects that vegetarian foods have high amount of copper compared to non-vegetarian foods.
10.4 Copper consumption and suggestions
Most nutritionists prescribe per day copper consumption in the ranges of 1 to 3mg; however, there is no assurance to this limit. Based on the research data, daily copper intake in the range of 4 to 7mg encouraged positive actions like reduction in harmful cellular oxidation along with lowering LDL level, and escalating HDL levels [11]. Nutritionists also proposed that the maximum limit should not be more than 10mg copper per day. Copper grants a defensive role in all circumstances with one omis­sion; copper should not be in daily use if one is affected by Wilson’s disorder. From the reported studies [18] we know that copper and zinc compete with each other for absorption in the body. Therefore, a higher amount of copper consumption can dimin­ish the zinc uptake and vice versa. Thus, a balance is always appreciated between these two metals ions for the betterment of human health. The recommended ratio of zinc and copper is 7:1. Moreover, one can enhance per day consumption of copper by eating copper-rich foods.
10.5 Mechanism of copper transport in humans
According to the recommendations, the minimal acceptable intake of copper for adult is about 0.9–1.3 mg per day, whereas the usual human being consumes more than that per day [20]. Intake of 2–3mg/day of copper is safe and avoids copper deficiency. However, in a normal person, intake of a higher amount of copper has an unimport­ant or no effect on its absorption because under normal conditions the amount of dietary copper that first enters into the stomach and is then absorbed in the upper intestinal tract is around 0.5mg/day which finally reaches the liver after forming com­plexes with proteins [21], and the remaining amount of copper is directly eliminated without entering pathways of absorption [22, 23]. Therefore, the liver is the major stockpile for intracellular copper [24]. This occurs mainly through the employment of metallothioneins that play a key role as a “mucosal block” for copper bioavail­ability. Copper is mainly transported across plasma membrane by CTR1 transporters and always intracellular copper remains in a complex form to prevent the oxidative damage to DNA, proteins and membrane components caused by free copper ions. Hence, copper transportation and use involves a complex interaction between trans­porters and binding proteins. Copper also plays a very important role as a key back­bone for catalytic centers in metalloenzymes named cofactors [25]. For a smooth and well-organized transportation and distribution of cupric for biological utility, a wide range of proteins play the key roles. The family of copper-bearing proteins imparts