Use of Solid Timber Waste as Potential Raw Material for Novel Herbal Drugs: Multidisciplinary Research, Development, and Innovation

Solid timber waste obtained from wood industries contains chemical compounds that may be used in phytochemical and pharmacological studies, and possess great potential for use in drug discovery. For its use as a raw material by the pharmaceutical, cosmeceutical, and veterinary industries, multidisciplinary studies and planning are needed. In this study, the wood of 3 trees, Willd. ( Fabaceae ), Bowdichia (Spr.) Benth ( Fabaceae ) and Myroxylon balsamum (L.) Harms ( Fabaceae ), was selected for phytochemical and pharmacological studies to evaluate antiproliferative activities in human tumor cells. The results obtained demonstrated the potential of solid wood residue a source of raw material for the discovery of pharmacologically active compounds. This source has been poorly explored, considering that it holds tremendous potential for the development of novel herbal drugs. However, it is necessary to implement a National Solid Waste Policy, proposing rules to improve management, classification and collection of solid waste generated by lumber based industries; which could allow a safe use of wood residues in phytochemical and pharmacological studies. The extracts studied demonstrate the potential to add value to non-timber forest products.


Obtaining the timber material
Discarded timber belonging to 3 woody species was obtained for phytochemical analyses from the wood flooring industry, Indus parquet Ltda. (Tietê, São Paulo). Only large solid timber waste that is discarded during the production of wooden flooring was used.

Identification of the timber samples
Macroscopic identification of the samples based on anatomy was performed by Professor Dr. João Perez Chimelo.

Preparation of the timber samples
Organic solvents used in this study were obtained from Merck (Sigma-Aldrich, São Paulo-Brasil). The previously identified and separated timber samples were processed by the Forestry Engineering Faculty, department of forest products (ESALQ/USP) and milled in a Wiley mill (mesh 10). The samples were subjected to Soxhlet extraction, initially with dichloromethane (a medium polarity organic solvent) yielding the dichloromethane crude extract (CDE), and later with absolute ethanol (a polar organic solvent) providing the ethanolic crude extract (EC. Solvents were evaporated from the DCE at 40 °C and from the ECE at 50 °C in a rotary evaporator, after which both the extracts were lyophilized.
Stock solutions of the samples (5mg) were prepared in dimethyl sulfoxide (DMSO, 50μL) followed by serial dilutions in RPMI/FBS 5% to give final concentrations of 0.25, 2.5, 25, and 250μg/mL. Doxorubicin was used as a positive control at the same concentrations. Cells seeded in 96-well plates (100μL/well, 3-7×10 4 cells/mL) were incubated, with each of the 4 concentrations of sample solution or doxorubicin (100μL/well) in triplicate (n = 3), for 48 h at 37 °C and 5% CO 2 . Before (T0 plate) and after (T1 plates) sample addition, cells were fixed with 50% trichloroacetic acid (50μL/well) and stained with sulforhodamine B to quantify cell proliferation based on the absorption values obtained using the Molecular Devices Versa Max Microplate Reader at 540nm. The Origin 8.0 software (Origin Lab Corporation) was used to evaluate the cytotoxic activity of the extracts. Samples were considered to possess antitumor activity when growth inhibition >50% and concentration-dependent cell-type selectivity was observed.

Results and Discussion
Phytochemical extraction of the collected timber samples was conducted, and the extracts were assayed for antiproliferative activities in human tumor cells. The in vitro assay results are expressed in terms of the sample concentration (μg/mL) and percentage growth of human tumor cells. A population growth of <50% indicated cytostatic action (inhibition of cell growth), whereas that <0% (a decline in cell population by cell death) indicated cytocidal activity. Doxorubicin was considered as a positive control for the antiproliferative activity on tumor cells, which exhibited a dose-dependent activity profile. At 2.5µg/mL, it exerted cytostatic activity on kidney, lung, prostate, melanoma, and leukemia cells; at 25μg/mL, it had cytostatic activity on colon, kidney, and ovarian cells, and cytocidal activity on melanoma, leukemia, and prostate cells. At the highest concentration (250µg/ mL), it had a cytostatic effect on cells of breast, kidney, and colon and ovarian cells expressing a multiple drug resistance phenotype; it had a cytocidal effect on the remaining cells at this concentration (Figure1).    Analysis of the concentration-response curve of the ECE of Bowdichia nitida ( Figure 3A) at 0.25µg/mL, demonstrated a cytostatic activity on prostate, breast, lung, and leukemia cell lines and a cytostatic profile activity only on the prostate cancer cell line. At 2.5μg/mL, the ECE was cytocidal to melanoma, leukemia, and prostate cancer cells; a cytostatic effect was observed on the other cell lines. At 250µg/mL, a cytocidal effect was observed on all cell lines. The DCE of Bowdichia nitida ( Figure 3B), at concentrations of 0.25µg/mL and 2.5µg/mL, a cytostatic activity was observed on leukemia and prostate cancer cells lines. At 25µg/mL, this extract demonstrated cytostatic activity on all but the leukemia cell line, on which it showed a cytocidal action. At 250μg/mL, the extract had a cytocidal profile, as it caused death of all the cells lines.

Mod Appl Pharm Pharmacol
and leukemia cells. At 250μg/mL, the extract showed a cytostatic activity on prostate cancer and breast cancer cell lines, and a cytocidal activity on all the other cell lines. The DCE of Myroxylon balsamum (Figure 4B), demonstrated a cytostatic activity at 0.25μg/mL on the breast cancer cells and at 2.5μg/mL on leukemia cells; and a cytocidal activity at 25µg/mL on kidney, lung, prostate, melanoma, breast, leukemia, and multiple drug resistant ovarian cells. In 250µg/mL, it demonstrated a cytostatic activity only on the ovarian cell line and a cytocidal activity on the remaining eight cell lines.
The timber used in this study is a source of several chemical compounds with antitumor potential, as demonstrated by our findings. Moreover, the use of timber discarded during manufacturing of forest products, such as wood used in construction and flooring, presents a significant advantage. It is estimated that approximately 10 million tons of these wastes are discarded annually in Brazil [16]. In this study, only large solid timber waste was used to aid anatomical identification. The majority industries do not have any plans for management of wood waste [17].
Bowdichia nitida , known as sucupira, is widely used in the wood industry. Due to its association with the development of contact allergy, studies on its chemical composition were carried out; the studies revealed the presence of 2,6-dimethoxy-1,4benzoquinone [18]. The ECE of Bowdichia nitida demonstrated cytocidal activity at 25μg/mL on the prostate cancer, leukemia, and melanoma cell lines. Selectivity for these 3 cell lines justifies further phytochemical and pharmacological studies for the possible isolation and identification of the active principles. Its DCE did not present any significant anticancer activity.
Wood from Dipteryx odorata, popularly known as cumaru, is commonly used in construction and flooring. Its extractives are used in the production of cigarettes, perfumes, and cosmetics [19]. The DCE of this species showed selective cytocidal activity at 25μg/mL on leukemia and ovarian cancer cell lines. Research reported that flavonoids in its chemical composition contribute to its anticancer activity [19].
Isolation of isoflavanoids, flavanones, and isoflavanones from the trunk and a pentacyclic triterpene from the leaves Myroxylon balsamum has been reported [20]. Results of the antiproliferative activity on tumor cells demonstrated that the DCE exhibited selective cytocidal activity at 25µg/mL on melanoma, colon, kidney, lung, breast, multiple drug resistant ovarian cancer, and leukemia cells.
However, the optimum use of plants is limited by the following challenges: 1) lack of laws regulating the extraction of plants, either for research or commercial purposes, leading to predatory exploitation and damage to the environment; 2) complexity of chemical structures, making it difficult to synthesize these compounds in the laboratory and elucidate their mechanisms of action; 3) time and cost associated with the isolation and synthesis of, pharmacotechnical processes involved, and preclinical, and clinical studies on the active principle isolated from a plant before its commercialization as a new drug; and 4) the amount of plant material needed to carry out the studies [12].
For solid wood waste to be used as raw material, it must be correctly identified according to the botanical classification of its native species. When obtained from industries as discarded material, collecting and identifying the plant species, based on the anatomy of wood, and cataloging of this process is crucial.
Currently, the National Solid Waste Policy (Policy No. 12,305) in Brazil defines the distinction between solid waste and discards [17,21]. According to the policy, timber industries must present a plan for the management of waste, discards, and by-products. Therefore, the use of solid wood waste as a source of raw material for pharmaceutical or cosmeceutical research and development is emphasized [22,23].

Conclusion
Solid timber residues, due to their chemical composition, are sources poorly explored by the chemical and pharmaceutical industries due the potential for the study of herbal medicine and new drugs. However, it is necessary to implement a National Solid Waste Policy so that solid waste can be managed during the manufacture of logs in the industries, classified and properly collected so that they are safely used in phytochemical and pharmacological studies. The residues studied demonstrate the potential to add value to non-timber forest products.